(19)
AUSTRALIAN PATENT OFFICE
(11) Application No. fr\J 2004297111 A1
(54)
Title
Anti-Mpl antibody
/^h1 ^ B
1° >)
International Patent Classification(s)
C07K. 16/28 (2006.01)20060101AFI2006040
C07K 16/28 8BMEP
PCT/JP2004/018506
(21)
Application No: 20042971 1 1
(22) Application Date: 2004.12.10
(87)
WIPO No: WO05/056604
(30)
(31 )
Priority Data
Number (321 Date
2004-248323 2004.00.27
2003- 415746 2003.12.12
2004- 071763 2004.03.12
(33) Country
JP
JP
JP
(43)
Publication Date : 2005.06.23
(71)
Applicant(s)
Chugai Seiyaku Kabushiki Kaisha
(72)
Inventor(s)
Tsuchiya. Masayukij Nakano. Kiyotaka;
Tsunoda. Hiroyuki; Hirata. Yuichi; Orita, Tetsuro
(74)
Agent/Attorney
Spruson & Ferguson. Level 35, St Martins Tower 31 Market Street Sydney. NSW. 2000
-1-
1
AU900W7III
VERIFICATION OF TRANSLATION
I, Mikiko Oyanagi
of the Shimizu Patent Office, 6th Floor., Kantetsu Tsukuba-Science-City Bldg. 1-1-1,
Oroshi-machi, Tsuchiura, Ibaraki, JAPAN 300-0847
declare as follows:
1 . That I am well acquainted with both the English and Japanese languages, and
2. That the attached document is a true and correct translation, made by me to the best of
my knowledge and belief, of:-
International Application No. PCT/JP2004/0 18506
(Publication No. WO2005/056604)
Entitled: " ANTI-MPL ANTIBODY "
Filed on December 10, 2004
Mo(y ' I , fc>
(Date)
(Signature of translator)
Mikiko Oyanagi
•179179*
I1N11IU
•G00002*
1
DESCRIPTION
ANT I -M PL ANTIBODY
Technical Field
5 The present invention relates to anti-Mpl antibodies.
Background Art
Thrombopoietin (TPO) is a factor that enhances the
differentiation and maturation of megakaryocytes (platelet precursor
10 cells) from hemopoietic stem cells into platelets. TPO also
functions as a cytokine with an important role in the regulation of
platelet number. TPO is converted into its active form through the
cleavage of a TPO precursor comprising 353 amino acids.
Mpl is a TPO receptor, and human Mpl molecules are known to exist
15 in two forms comprising 572 and 635 amino acids. The human Mpl gene
sequence has already been analyzed (see Non-Patent Document 1 and
GenBank accession No. NM_005373) .
Most cytokine receptors dimerize upon ligand binding, and
transduce signals into cells. • It has been reported that TPO similarly
20 binds to its own specific receptor MPL, which leads to dimerization
of the receptor, thereby transducing signals into cells and exerting
physiological effects (see Non-Patent Document 2) .
Antibodies exhibiting agonistic activity have been reported
among those antibodies that bind to receptors having the above
25 features.
For example, an antibody against the erythropoietin (EPO)
receptor has been reported to substitute for erythropoietin function.
The monovalent form (Fab) of the antibody is capable of binding to
the EPO receptor but is unable to transduce signals. Thus,
30 dimerization of the erythropoietin receptor via bivalent binding is
assumed to be essential for signal transduction (see Non-Patent
Document 3) .
Antibodies that bind to Mpl and exhibit TPO agonistic activity
have also been reported (see Non-Patent Documents 4 and 5) . This
35 suggests that receptor dimerization is induced upon binding of a
bivalent antibody with regards to MPL as well.
2
Meanwhile, a single-chain antibody (scFv) has been reported to
exhibit TPO agonistic activity (see Patent Document 1) . However, it
has been revealed that, the underlying mechanism of scFv exhibiting
TPO agonistic activity is that a part of scFv diraerizes (diabody)
5 and this diabody becomes the actual active unit (see Patent Documents
2 to 4) .
[Patent Document 1] US Patent No. 6342220
[Patent Document 2] WO 01/79494
[Patent Document 3] WO 02/33072
10 [Patent Document 4] WO 02/33073
[Non-Patent Document 1] Palacios et al., Cell, 1985, 41, 727-734
[Non-Patent Document 2] Souyri et al., Cell, 1990, 63, 1137-1147
[Non-Patent Document 3] Elliott, S. et al., J. Biol. Chem., 1996,
271(40), 24691-24697
15 [Non-Patent Document 4] Abe et al., Immunol. Lett., 1998, 61, 73-78
[Non-Patent Document 5] Bi j ia Deng et al . , Blood, 1998, 92, 1981-1988
Disclosure of the Invention
Problems to Be Solved by the Invention
20 The present invention was achieved in view of the above
circumstances. An objective of the present invention is to provide
novel anti-Mpl antibodies having TPO agonistic activity.
Means to Solve the Problems
25 The present inventors performed exhaustive research to solve
the above objective. The present inventors prepared and purified
anti-human Mpl antibody VB22B, and established a single-chain
antibody expression system using genetic engineering techniques.
Specifically, the variable region of anti-human Mpl antibody was first
30 cloned, and a diabody expression vector pCXND3-VB22B db for the
anti-human Mpl antibody was prepared. This pCXND3-VB22B db vector
was then used to generate an expression vector pCXND3-VB22B sc(Fv)2
for anti-human Mpl antibody sc(Fv)2. Anti-human Mpl sc(Fv)2 was
expressed in CHO-DG44 cells using the expression vector pCXND3-VB22B
35 sc(Fv)2, and then purified from the culture supernatant. In control
experiments, VB22B diabody was transiently expressed in COS7 cells
3
using the above pCXND3-VB22B db vector, and then purified from the
culture supernatant.
In addition, VB22B diabody and VB22B sc (Fv) 2 were evaluated for
their TPO-like agonistic activities. The results showed that VB22B
5 diabody and VB22B sc(Fv)2 exhibit higher agonistic activities
compared to VB22B IgG, and thus activities equivalent to or higher
than that of the natural ligand, human TPO.
Furthermore, the present inventors succeeded in preparing five
types of humanized VB22B sc(Fv)2. The TPO-like agonistic activity
10 was also proven to be unaltered by humanization.
More specifically, the present invention provides the following
(1) to (38):
(1) an antibody comprising a single-chain polypeptide having
binding activity against TPO receptor (Mpl), wherein said antibody
15 comprises two heavy chain variable regions and two light chain
variable regions;
(2) the antibody of (1), wherein the two heavy chain variable
regions and the two light chain variable regions are arranged in the
order of heavy chain variable region, light chain variable region,
20 heavy chain variable region, and light chain variable region from
the N terminus of the single-chain polypeptide;
(3) the antibody of (1) or (2), wherein the two heavy chain
variable regions and the two light chain variable regions are linked
by linkers;
25 (4) the antibody of (3), wherein the linkers comprise 15 amino
acids;
(5) a chimeric antibody that binds to Mpl;
(6) the antibody of (5), which is a humanized antibody;
(7) the antibody of (5) or (6), which is a minibody;
30 (8) an antibody that binds to soluble Mpl;
(9) an antibody that binds to human Mpl and monkey Mpl;
(10) an antibody having agonistic activity against human Mpl
and monkey Mpl;
(11) an antibody whose binding activity to soluble Mpl is KD
35 = 10" 6 M or lower;
(12) an antibody whose binding activity to soluble Mpl is KD
4
= 10 7 M or lower;
(13) an antibody whose TPO agonistic activity is EC50 =100 nM
or lower;
(14) an antibody whose TPO agonistic activity is EC50 = 30 nM
5 or lower;
(15) an antibody whose TPO agonistic activity is EC50 = 10 nM
or lower;
(16) an antibody which comprises a heavy chain variable region,
wherein said heavy chain variable regions comprises CDR1, CDR2 and
10 CDR3 consisting of an amino acid sequence of any one of:
[1) SEQ ID NOs: 3, 4, and 5
[2] SEQ ID NOs: 6, 7, and 8
[3] SEQ ID NOs: 9, 10, and 11
14] SEQ ID NOs: 15, 16, and 17
15 [5] SEQ ID NOs: 18, 19, and 20
[6] SEQ ID NOs: 21, 22, and 23
[7] SEQ ID NOs: 24, 25, and 26
[8] SEQ ID NOs: 27, 28, and 29
[9] SEQ ID NOs: 30, 31, and 32
20
[10]
SEQ
ID
NOs:
33,
34,
and
35
[11]
SEQ
ID
NOs:
36,
37,
and
38
[12]
SEQ
ID
NOs :
39,
40,
and
41
[13]
SEQ
ID
NOs:
42,
43,
and
44
[14]
SEQ
ID
NOs :
48,
49,
and
50
25
[15]
SEQ
ID
NOs :
51,
52,
and
53
[16]
SEQ
ID
NOs :
54,
55,
and
56
[17]
SEQ
ID
NOs:
57,
58,
and
59;
(17) an antibody which comprises a light chain variable region,
wherein said light chain variable region comprises CDRl, CDR2 and
30 CDR3 consisting of an amino acid sequence of any one of:
[1] SEQ ID NOs: 60, 61, and 62
[2] SEQ ID NOs: 63, 64, and 65
[3] SEQ ID NOs: 78, 79, and 80
[4] SEQ ID NOs: 84, 85, and 86
35 [5] SEQ ID NOs: 93, 94, and 95
[6] SEQ ID NOs: 96, 97, and 98
[7] SEQ ID NOs: 102, 103, and 104
[8] SEQ ID NOs : 108, 109, and 110
[9] SEQ ID NOs: 111, 112, and 113
[10] SEQ ID NOs: 114, 115, and 116;
(IB) an antibody that comprises a heavy chain variable region
and a light chain variable region of any one of:
[1] a heavy chain variable region that comprises CDR1, CDR2 ,
and CDR3 comprising the amino acid sequences consisting of SEQ ID
NOs: 3, 4, and 5, and a light chain variable region that comprises
CDR1, CDR2, and CDR3 comprising the amino acid sequences consisting
of SEQ ID NOs: 60, 61, and 62;
[2] a heavy chain variable region that comprises CDR1, CDR2,
and CDR3 comprising the amino acid sequences consisting of SEQ ID
NOs: 6, 7, and 8, and a light chain variable region that comprises
CDRl, CDR2, and CDR3 comprising the amino acid sequences consisting
of SEQ ID NOs: 63, 64, and 65;
[3] a heavy chain variable region that comprises CDRl, CDR2,
and CDR3 comprising the amino acid sequences consisting of SEQ ID
NOs: 9, 10, and 11, and a light chain variable region that comprises
CDRl, CDR2, and CDR3 comprising the amino acid sequences consisting
of SEQ ID NOs: 63, 64, and 65;
[4] a heavy chain variable region that comprises CDRl, CDR2 ,
and CDR3 comprising the amino acid sequences consisting of SEQ ID
NOs: 15, 16, and 17, and a light chain variable region that comprises
CDRl, CDR2, and CDR3 comprising the amino acid sequences consisting
of SEQ ID NOs: 63, 64, and 65;
[5] a heavy chain variable region that comprises CDRl, CDR2,
and CDR3 comprising the amino acid sequences consisting of SEQ ID
NOs: 18, 19, and 20, and a light chain variable region that comprises
CDRl, CDR2, and CDR3 comprising the amino acid sequences consisting
of SEQ ID NOs: 63, 64, and 65;
[6] a heavy chain variable region that comprises CDRl, CDR2,
and CDR3 comprising the amino acid sequences consisting of SEQ ID
NOs: 21, 22, and 23, and a light chain variable region that comprises
CDRl, CDR2, and CDR3 comprising the amino acid sequences consisting
of SEQ ID NOs: 78, 79, and 80;
6
[7] a heavy chain variable region that comprises CDR1, CDR2,
and CDR3 comprising the amino acid sequences consisting of SEQ ID
NOs : 24, 25, and 26, and a light chain variable region that comprises
CDR1, CDR2, and CDR3 comprising the amino acid sequences consisting
5 of SEQ ID NOs: 63, 64, and 65;
[8] a heavy chain variable region that comprises CDR1, CDR2,
and CDR3 comprising the amino acid sequences consisting of SEQ ID
NOs: 27, 28, and 29, and a light chain variable region that comprises
CDR1, CDR2, and CDR3 comprising the amino acid sequences consisting
10 of SEQ ID NOs: 84, 85, and 86;
[9] a heavy chain variable region that comprises CDR1, CDR2,
and CDR3 comprising the amino acid sequences consisting of SEQ ID
NOs: 30, 31, and 32, and a light chain variable region that comprises
CDR1, CDR2, and CDR3 comprising the amino acid sequence consisting
15 of SEQ ID NOs: 63, 64, and 65;
[10] a heavy chain variable region that comprises CDR1, CDR2,
and CDR3 comprising the amino acid sequences consisting of SEQ ID
NOs: 33, 34, and 35, and a light chain variable region that comprises
CDR1, CDR2, and CDR3 comprising the amino acid sequences consisting
20 of SEQ ID NOs: 63, 64, and 65;
[11] a heavy chain variable region that comprises CDR1, CDR2,
and CDR3 comprising the amino acid sequences consisting of SEQ ID
NOs: 36, 37, and 38, and a light chain variable region that comprises
CDR1, CDR2, and CDR3 comprising the amino acid sequences consisting
25 of SEQ ID NOs: 93, 94, and 95;
[12] a heavy chain variable region that comprises CDR1, CDR2,
and CDR3 comprising the amino acid sequences consisting of SEQ ID
NOs: 39, 40, and 41, and a light chain variable region that comprises
CDR1, CDR2, and CDR3 comprising the amino acid sequences consisting
30 of SEQ ID NOs: 96, 97, and 98;
[13] a heavy chain variable region that comprises CDR1, CDR2,
and CDR3 comprising the amino acid sequences consisting of SEQ ID
NOs: 42, 43, and 44, and a light chain variable region that comprises
CDR1, CDR2, and CDR3 comprising the amino acid sequences consisting
35 of SEQ ID NOs: 78, 79, and 80;
[14] a heavy chain variable region that comprises CDR1, CDR2,
7
and CDR3 comprising the amino acid sequences consisting of SEQ ID
NOs: 45, 46, and 47, and a light chain variable region that comprises
CDR1, CDR2, and CDR3 comprising the amino acid sequences consisting
of SEQ ID NOs: 102, 103, and 104;
5 [15] a heavy chain variable region that comprises CDR1, CDR2,
and CDR3 comprising the amino acid sequences consisting of SEQ ID
NOs: 48, 49, and 50, and a light chain variable region that comprises
CDR1, CDR2, and CDR3 comprising the amino acid sequences consisting
of SEQ ID NOs: 63, 64, and 65;
10 [16] a heavy chain variable region that comprises CDR1, CDR2,
and CDR3 comprising the amino acid sequences consisting of SEQ ID
NOs: 51, 52, and 53, and a light chain variable region that comprises
CDR1, CDR2, and CDR3 comprising the amino acid sequences consisting
of SEQ ID NOs: 108, 109, and 110,
15 [17] a heavy chain variable region that comprises CDR1, CDR2,
and CDR3 comprising the amino acid sequences consisting of SEQ ID
NOs: 54, 55, and 56, and a light chain variable region that comprises
CDR1, CDR2, and CDR3 comprising the amino acid sequences consisting
of SEQ ID NOs: 111, 112, and 113;
20 [18] a heavy chain variable region that comprises CDR1, CDR2,
and CDR3 comprising the amino acid sequences consisting of SEQ ID
NOs: 57, 58, and 59, and a light chain variable region that comprises
CDR1, CDR2, and CDR3 each comprising the amino acid sequences
consisting of SEQ ID NOs: 114, 115, and 116;
25 (19) an antibody that comprises a heavy chain variable region
comprising the amino acid sequence of SEQ ID NO: 118;
(20) an antibody that comprises a light chain variable region
comprising the amino acid sequence of SEQ ID NO: 120;
(21) an antibody that comprises a heavy chain variable region
30 comprising the amino acid sequence of SEQ ID NO: 118 and a light chain
variable region comprising the amino acid sequence of SEQ ID NO: 120;
(22) an antibody comprising the amino acid sequence of SEQ ID
NO: 122 or 264;
(23) an antibody that comprises a heavy chain variable region,
35 wherein said heavy chain variable region comprises FR1, FR2, FR3,
and FR4 consisting of amino acid sequences of any one of:
8
[1]
SEQ
ID NOs: 230, 232, 234,
and 236
[2]
SEQ
ID NOs: 265, 267, 269,
and 271
[3]
SEQ
ID NOs: 279, 281, 283,
and 285
[4]
SEQ
ID NOs: 298, 299, 300,
and 301
[5]
SEQ
ID NOs: 298, 299, 306,
and 301.
(24)
an
antibody comprising a
light chain variable region,
wherein said light chain variable region comprises FR1, FR2, FR3,
and FR4 consisting of amino acid sequences of any one of:
[1] SEQ ID NOs: 239, 241, 243, and 245
10 [2] SEQ ID NOs: 272, 274, 276, and 278
[3] SEQ ID NOs: 302, 303, 304, and 305
[4] SEQ ID NOs: 302, 307, 308, and 305;
(25) an antibody that comprises a heavy chain variable region
and a light chain variable region of any one of:
15 [1] a heavy chain variable region which comprises FR1, FR2, FR3,
and FR4 having the amino acid sequences consisting of SEQ ID NOs:
230, 232, 234, and 236, and a light chain variable region which
comprises FR1, FR2, FR3, and FR4 having the amino acid sequences
consisting of SEQ ID NOs: 239, 241, 243, and 245;
20 [2] a heavy chain variable region which comprises FR1 , FR2, FR3,
and FR4 having the amino acid sequences consisting of SEQ ID NOs:
265, 267, 269, and 271, and a light chain variable region which
comprises FR1, FR2, FR3, and FR4 having the amino acid sequences
consisting of SEQ ID NOs: 272, 274, 276, and 278;
25 [3] a heavy chain variable region which comprises FR1 , FR2 , FR3,
and FR4 having the amino acid sequences consisting of SEQ ID NOs:
279, 281, 283, and 285, and a light chain variable region which
comprises FR1, FR2, FR3, and FR4 having the amino acid sequences
consisting of SEQ ID NOs: 272, 274, 276, and 278;
30 [4] a heavy chain variable region which comprises FR1, FR2, FR3,
and FR4 having the amino acid sequences consisting of SEQ ID NOs:
298, 299, 300, and 301, and a light chain variable region which
comprises FR1, FR2, FR3, and FR4 having the amino acid sequences
consisting of SEQ ID NOs: 302, 303, 304, and 305;
35 [5] a heavy chain variable region which comprises FR1, FR2, FR3,
and FR4 having the amino acid sequences consisting of SEQ ID NOs:
9
298, 299, 306, and 301,' and a light chain variable region which
comprises FR1, FR2 , FR3, and FR4 having the amino acid sequences
consisting of SEQ ID NOs: 302, 307, 308, and 305;
(26) an antibody that comprises a heavy chain variable region,
5 wherein said heavy chain variable region comprises the amino acid
sequence of SEQ ID NO: 229, 256, 262, 289, or 295;
(27) an antibody that comprises a light chain variable region,
wherein said light chain variable region comprises the amino acid
sequence of SEQ ID NO: 238, 258, 291, or 297;
10 (28) -an antibody that comprises a heavy chain variable region
and a light chain variable region of any one of:
[1] a heavy chain variable region comprising the amino acid
sequence of SEQ ID NO: 229, and a light chain variable region
comprising the amino acid sequence of SEQ ID NO: 238;
15 [2] a heavy chain variable region comprising the amino acid
sequence of SEQ ID NO: 256, and a light chain variable region
comprising the amino acid sequence of SEQ ID NO: 258;
[3] a heavy chain variable region comprising the amino acid
sequence of SEQ ID NO: 262, and a light chain variable region
20 comprising the amino acid sequence of SEQ ID NO: 258;
[4] a heavy chain variable region comprising the amino acid
sequence of SEQ ID NO: 289, and a light chain variable region
comprising the amino acid sequence of SEQ ID NO: 291;
[5] a heavy chain variable region comprising the amino acid
25 sequence of SEQ ID NO: 295, and a light chain variable region
comprising the amino acid sequence of SEQ ID NO: 297;
(29) an antibody that comprises the amino acid sequence of SEQ
ID NO: 2, 254, 260, 287, or 293;
(30) an antibody having an activity equivalent to that of an
30 antibody of any one of (16) to (29), wherein said antibody comprises
the amino acid sequence set forth in any one of (16) to (29) , in which
one or more amino acids have been substituted, deleted, added and/or
inserted;
(31) an antibody that recognizes an epitope recognized by an
35 antibody of any one of (16) to (30);
(32) an antibody that recognizes the region of amino acids. 26
10
to 27 4 of human Mpl;
(33) an antibody of any one of (1) to (32), which has TPO
agonistic activity;
(34) a polynucleotide encoding an antibody of any one of (1)
to (33) ;
(35) a polynucleotide hybridizing to the polynucleotide of (34)
under stringent conditions, wherein said polynucleotide encodes an
antibody having activity equivalent to that of an antibody of any
one of (1) to (33) ;
(36) a vector comprising the polynucleotide of (34) or (35);
(37) a host cell that carries the polynucleotide of (34) or (35) ,
or the vector of (36) ; and
(38) a pharmaceutical composition comprising an antibody of any
one of (1) to (33') .
Brief Description of the Drawings
Fig. 1 demonstrates the strategy for preparing single-chain
antibody sc(Fv)2.
Fig. 2 illustrates the assessment of VB22B sc(Fv)2 binding
activity using an Mpl-expressing CHO cell line. Purified VB22B
sc(Fv)2 was used.
Fig. 3 illustrates the assessment of VB22B antibody agonistic
activity using BaF3-human Mpl.
Fig. 4 illustrates the assessment of VB22B antibody agonistic
activity using BaF3-monkey Mpl.
Fig. 5 illustrates the assessment of VB22B antibody agonistic
activity using M-07e.
Fig. 6 shows the amino acid sequences of anti-human Mpl
antibodies (H chains) that exhibit higher agonistic activities when
converted into minibodies.
Fig. 7 shows the amino acid sequences of anti-human Mpl
antibodies (L chains) which exhibit higher agonistic activities when
converted into minibodies.
Fig. 8 illustrates the binding activity assessment of AB317
diabody using Mpl-expressing CHO cells. Both VB22B diabody (solid
line) and AB317 diabody (broken line) were obtained from C0S7 culture
11
supernatants .
Fig. 9 illustrates the agnostic activity assessment of AB324
and AB317 diabodies using BaF3-human Mpl .
Fig. 10 illustrates the agnostic activity assessment of AB324
and AB317 diabodies using BaF3-monkey Mpl.
Fig. 11 illustrates the agnostic activity assessment of AB324
and AB317 diabodies using BaF3-mouse Mpl.
Fig. 12 shows the agonistic activities of diabodies in
BaF3-human Mpl cells. The X-axis shows OD at 450/655 rati, and the
Y-axis represents concentration.
Fig. 13 shows the agonistic activities of diabodies in
BaF3-human Mpl (G305C) cells. The X-axis shows OD at 450/655 nm, and
the Y-axis represents concentration.
Fig. 14 shows the agonistic activities of TA136 db and TA136
sc(Fv)2 in 8aF3-human Mpl cells. The X-axis shows OD at 450/655 nm
and the Y-axis represents concentration.
Fig. 15 shows the agonistic activities of TA136 db and TA136
sc(Fv)2 in BaF3-human Mpl (G305C) cells. The X-axis shows OD at
450/655 nm, and the Y-axis represents concentration.
Fig. 16 shows the agonistic activities of TA136 db and TA136
sc{Fv)2 in BaF3-human Mpl (C769T) cells. The X-axis shows OD at
450/655 nm, and the Y-axis represents concentration.
Fig. 17 shows the agonistic activities of TA136 db and TA136
sc(Fv)2 in BaF3-human Mpl (C823A) cells. The X-axis shows OD at
450/655 nm, and the Y-axis represents concentration.
Fig. 18 shows the positions of FRs and CDRs in humanized heavy
chain seguences (hVB22B p-z, hVB22B g-e, hVB22B e, hVB22B u2-wz4,
and hVB22B q-wz5:VH), and humanized light chain sequences (hVB22B
p-z, hVB22B g-e, hVB22B e, hVB22B u2-wz4, and hVB22B q-wz5:VL).
Fig. 19 shows the TPO-like agonistic activities of murine VB22B
sc(Fv)2, hVB22B e sc(Fv)2, and hVB22B g-e sc(Fv)2 in BaF3-human Mpl.
The X-axis shows absorbance ratio (450 nm/655 nm) , and the Y-axis
represents concentration.
Fig. 20 shows the TPO-like agonistic activities of murine VB22B
sc(Fv)2, hVB22B p-z sc (Fv) 2, and hVB22B u2-wz4 sc(Fv)2in BaF3-human
Mpl. The X-axis shows absorbance ratio {450 nm/655 nm) , and the Y-axis
12
represents concentration.
Fig. 21 shows the TPO-like agonistic activities of murine VB22B
sc(Fv}2 and hVB22B q-wz5 sc(Fv)2 in BaF3-human Mpl . The X-axis shows
absorbance ratio (450 nm/655 nm) , and the Y-axis represents
5 concentration.
Best Mode for Carrying Out the Invention
The present invention provides antibodies that bind to the TPO
receptor (Mpl) .
10 The antibodies of the present invention comprise all types of
antibodies, including antibodies with modified amino acid sequences,
such as minibodies, humanized antibodies, and chimeric antibodies;
antibodies that have been modified by binding with other molecules
(for example, polymers such as polyethylene glycol) ; and antibodies
15 whose sugar chains have been modified.
Mpl of the present invention may be a mutant receptor. A mutant
receptor of the present invention is usually a receptor that exists
at a frequency lower than 50%, preferably lower than 20%, more
preferably lower than 10%, and even more preferably lower than 1%.
20 The frequency is generally calculated using randomly selected
subjects. However, the frequency may vary depending on the country,
area, sex, and such. Therefore, the frequency may also be calculated,
for example, within a defined country or area, such as Japan, the
United States, and Europe, or calculated for one sex. When there are
25 two or more mutations in a receptor, the frequency may be calculated
for multiple mutation sites or for any one of the mutation sites.
Mutant receptors are preferably evaluated by a frequency as described
above. However, mutant receptors can also be evaluated, for example,
by their signal transducing ability and such. Specifically, for
30 example, when two different receptors are present, the one with
stronger transducing signals upon natural ligand-binding maybe be
used as a non-mutant type receptor, and the one with weaker transducing
signals as a mutant receptor.
In one embodiment, the mutant receptors of the present invention
35 comprise receptors that are associated with disease onset. The
phrase "mutant receptors associated with disease onset" means that
13
the loss of reactivity to a natural ligand becomes part of the reason
that triggers disease onset. In the present invention, the mutant
receptor may be a contributing factor, but not necessarily the sole
factor triggering disease onset. Many reports have been previously
5 published that describe the association of mutant receptors with
disease onset. In addition to those that have been reported,
associations of mutant receptors and disease onset can also be
identified by statistical analysis methods (for example, correlation
analyses) . Correlation analyses, also called "case control studies",
10 are well known to those skilled in the art (for example, Nishimura,
Y . , 1991, "Statistical analysis of polymorphisms", Saishin Igaku,
46:909-923; Oka, A. et al . , 1990, Hum. Mol. Genetics 8, 2165-2170;
Ota, M. et al., 1999, Am. J. Hum. Genet. 64, 1406-1410; Ozawa, A.
et al., 1999, Tissue Antigens 53, 263-268). For example, the
15 correlation between a mutant receptor and a disease can be studied
by computing the freguency of the mutant receptor in patients and
healthy subjects, and testing whether the patient population has a
higher mutant receptor freguency. Typically, differences in
frequency are evaluated using the fc-test. X is obtained by the
20 equation % 2 - 2 (observed value - expected value ) 2 /expected value. A
p value is obtained from the X 2 value determined. Based on this p
value, it can be determined whether there is a correlation between
the mutant receptor and the disease. For example, when p<0.05, the
mutant receptor is considered to correlate with the disease. Mutant
25 thrombopoietin (TPO) receptors have already been reported (Matthias
Ballmaier et al., 2001, BLOOD, 97 (1), 139; and others).
It is preferable that the antibodies of the present invention
have agonistic activity against Mpl .
In a preferred embodiment, the antibodies of the present
30 invention comprise, for example, minibodies.
The minibodies comprise antibody fragments lacking portions of
the whole antibody (for example, whole IgG) . The minibodies are not
particularly limited as long as they have binding activity to their
antigens. The minibodies of the present invention have' higher
35 activities compared to their corresponding whole antibodies. There
are no particular limitations on the antibody fragments of the present
14
invention as long as they are portions of the whole antibody, and
preferably contain heavy chain variable regions (VH) and/or light
chain variable regions (VL) . The amino acid sequences of VH or VL
may contain substitutions, deletions, additions and/or insertions.
5 Furthermore, the antibody fragment may also lack portions of VH or/and
VL, as long as it has binding ability to its antigen. In addition,
the variable regions may be chimerized or humanized. Such antibody
fragments include, for example, Fab, Fab', F(ab')z, and Fv. An
example of a minibody includes Fab, Fab', F(ab') 2 , Fv, scFv
10 (single-chain Fv) , diabody, and sc(Fv)2 (single-chain (Fv)2).
Herein, an "Fv" fragment is the smallest antibody fragment and
contains a complete antigen recognition site and a binding site. The
"Fv" fragment is a dimer (VH-VL dimer) in which a single VH and a
single VL are strongly linked by a non-covalent bond. The three
15 complementarity-determining regions (CDRs) of each of the variable
regions interact with each other to form an antigen-binding site on
the surface of the VH-VL dimer. Six CDRs confer the antigen-binding
site of an antibody. However, a single variable region (or a half
of Fv containing only three CDRs specific to an antigen) alone is
20 also capable of recognizing and binding an antigen although its
affinity is lower than the affinity of the entire binding site.
scFv contains the VH and VL regions of an antibody, and these
regions exist on a single polypeptide chain. Generally, an Fv
polypeptide further contains a polypeptide linker between VH and VL,
25 and therefore an scFv can form a structure required for antigen binding .
See, Pluckthun "The Pharmacology of Monoclonal Antibodies" Vol. 113
(Rosenburg and Moore eds. (Springer Verlag, New York, pp. 269-315,
1994) for the review of scFv. In the present invention, linkers are
not especially limited as long as they do not inhibit expression of
30 antibody variable regions linked at both ends of the linkers.
The term "diabody" refers to a bivalent antibody fragment
constructed by gene fusion (Holliger P et al . , 1993, Proc. Natl . Acad.
Sci. USA 90: 6444-6448; EP 404,097; WO 93/11161 and others).
Diabodies are dimers comprising two polypeptide chains, where each
35 polypeptide chain comprises a VL and a VH connected with a linker
short enough to prevent interaction of these two domains, for example,
15
a linker of about five residues. The VL and VH encoded on the same
polypeptide chain will form a dimer because the linker between them
is too short to form a single-chain variable region fragment. As a
result, the polypeptide chains form a dimer, and thus the diabody
5 has two antigen binding sites.
sc (Fv) 2 is a single-chain minibody produced by linking two units
of VH and two units of VL with linkers and such (Hudson et ai., 1999,
J Immunol. Methods 231:177-189). sc(Fv)2 exhibits a particularly
high agonistic activity compared to the whole antibody and other
10 minibodies. sc (Fv) 2 can be produced, for example, by linking two scFv
molecules .
In a preferable antibody, the two VH units and two VL units are
arranged in the order of VH, VL, VH, and VL
( [VH] -linker- [VL] -linker- [VH] -linker- [VL] ) beginning from the N
15 terminus of a single-chain polypeptide.
The order of the two VH units and two VL units is not limited
to the above arrangement, and they may be arranged in any order.
Examples of the arrangements are listed below.
[VL] -linker- [VH] -linker- [VH] -linker- [VL]
2 0 [VH] -linker- [VL] -linker- [VL] -linker- [VH]
[VH] -linker- [VH] -linker- [VL] -linker- [VL]
[VL] -linker- [VL] -linker- [VH] -linker- [VH]
[VL] -linker- [VH] -linker- [VL] -linker- [VH]
The linkers to be used for linking the variable regions of an
25 antibody comprise arbitrary peptide linkers that can be introduced
by genetic engineering, synthetic linkers, and linkers disclosed in,
for example, Holliger, P. et al . , Protein Engineering, 9(3), 299-305,
1996. Peptide linkers are preferred in the present invention. There
are no limitations as to the length of the peptide linkers. The length
30 can be selected accordingly by those skilled in the art depending
on the purpose, and is typically 1-100 amino acids, preferably 3-50
amino acids, more preferably 5-30 amino acids, and even more
preferably 12-18 amino acids (for example, 15 amino acids) .
For example, such peptide linkers include:
35 Ser
Gly • Ser
16
GlyGly Ser
Ser-Gly ■ Gly
Gly Gly Gly Ser
Ser- Gly Gly Gly
5 Gly Gly Gly Gly Ser
Ser • Gly Gly • Gly Gly
Gly Gly Gly Gly Gly Ser
Ser • Gly Gly Gly Gly Gly
Gly Gly Gly • Gly Gly Gly Ser
10 Ser-Gly Gly GlyGly Gly Gly
(GlyGly Gly Gly Ser) „
(Ser-Gly-Gly Gly-Gly) n
where n is. an integer of 1 or larger. The lengths and sequences of
peptide linkers can be selected accordingly by those skilled in the
15 art depending on the purpose.
In an embodiment of the present invention, a particularly
preferable sc(Fv)2 includes, for example, the sc(Fv)2 below.
[VH] -peptide linker (15 amino acids) - [VL] -peptide linker (15 amino
acids) - [VH] -peptide linker (15 amino acids ) - [VL]
20 Synthetic linkers (chemical crosslinking agents) include
crosslinking agents routinely used to crosslink peptides, for example,
N-hydroxy succinimide (NHS), disuccinimidyl suberate (DSS),
bis ( succinimidyl ) suberate (BS 3 ) , dithiobis ( succinimidyl
propionate) (DSP), dithiobi s ( succinimidyl propionate) (DTSSP) ,
25 ethylene glycol bis ( succinimidyl succinate) (EGS) , ethylene glycol
bis ( sulf osuccinimidyl succinate) (sulfo-EGS), disuccinimidyl
tartrate (DST), disulf osuccinimidyl tartrate (sulfo-DST) ,
bis [ 2- ( succinimidoxycarbonyloxy) ethyl ] sulf one (BSOCOES) , and
bis [ 2- ( succinimidoxycarbonyloxy) ethyl ] sulfone (sulf o-BSOCOES) .
30 These crosslinking agents are commercially available.
In general, three linkers are required to link four antibody
variable regions together. The linkers to be used may be of the same
type or different types. In the present invention, a preferable
minibody is a diabody, even more preferably, an sc(Fv)2. Such a
35 minibody can be prepared by treating an antibody with an enzyme, for
example, papain or pepsin, to generate antibody fragments, or by
17
constructing DNAs encoding those antibody fragments and introducing
there into expression vectors, followed by expression in an appropriate
host cell (see, for example, Co, M. S. et al., 1994, J. Immunol. 152,
2968-2976; Better, M. and Horwitz, A. H., 1989, Methods Enzymol. 178,
476-496; Pluckthun, A. andSkerra, A., 1989, Methods Enzymol. 178,
497-515; Lamoyi, E . , 1986, Methods Enzymol. 121, 652-663; Rousseaux,
J. et al. , 1986, Methods Enzymol . 121, 663-669; Bird, R. E. and Walker,
B. W., 1991, Trends Biotechnol. 9, 132-137).
An antibody having exceedingly high agonistic activity can be
prepared by reducing the molecular weight of a full-length antibody,
particularly by converting it into an sc(Fv)2.
In a preferred embodiment, the antibodies of the present
invention comprise modified antibodies, such as chimeric antibodies
and humanized antibodies that bind to Mpl . These modified antibodies
can be produced by known methods.
Chimeric antibodies are antibodies prepared by combining
sequences derived from different animal species, and include for
example, antibodies comprising the heavy chain and light chain
variable regions of a murine antibody, and the heavy chain and light
chain constant regions of a human antibody. Chimeric antibodies can
be prepared by known methods . For example, a DNA encoding the V region
of an antibody is linked to a DNA encoding the C region of a human
antibody, and the construct is inserted into an expression vector
and introduced into a host to produce chimeric antibodies.
Humanized antibodies are also referred to as "reshaped human
antibodies". Such a humanized antibody is obtained by transferring
the complementarity-determining region (CDR) of an antibody derived
from a non-human mammal, for example mouse, to the
complementarity-determining region of a human antibody, and the
general gene recombination procedure for this is also known (see
European Patent Application No. 125023 and WO 96/02576) .
Specifically, a DNA sequence designed to link a murine antibody
CDR to the framework region (FR) of a human antibody can be synthesized
by PCR, using primers prepared from several oligonucleotides
containing overlapping portions of both CDR and FR terminal regions
(see methods described in WO 98/13388) .
18
The human antibody framework region to be linked by CDR is
selected in order to form a favorable antigen-binding site in the
complementarity-determining region. Amino acids of the framework
region in the antibody variable region may be substituted, as
5 necessary, for the complementarity-determining region of the reshaped
human antibody to form a suitable antigen-binding site (Sato, K. et
al., 1993, Cancer Res. 53, 851-856).
The constant region of a human antibody is used as the constant
region of a chimeric antibody or humanized antibody. For example,
10 Cyl, Cy2, Cy3, and Cy4 can be used as the H chain, and Ck and CX. can
be used as the L chain. The human antibody constant region may be
modified to improve the antibody or the stability of the antibody
production .
Generally, chimeric antibodies comprise. the variable region of
15 an antibody from a non-human mammal and the constant region derived
from a human antibody. On the other hand, humanized antibodies
comprise the complementarity-determining region of an antibody from
a non-human mammal, and the framework region and constant region
derived from a human antibody.
20 In addition, after a chimeric antibody or a humanized antibody
is prepared,' amino acids in the variable region (for example, FR)
and the constant region may be replaced with other amino acids, and
such .
The origin of the variable regions in chimeric antibodies or
25 that of the CDRs in humanized antibodies is not particularly limited,
and may be derived from any type of animal. For example, sequences
of murine antibodies, rat antibodies, rabbit antibodies, camel
antibodies may be used.
In general, it is difficult to chimerize or humanize an antibody
30 without losing the agonistic activity of the original antibody.
Nevertheless, the present invention succeeded in preparing humanized
antibodies having agonistic activity equivalent to that of the
original murine antibody.
A preferred humanized antibody of the present invention is an
35 antibody comprising a heavy chain variable region that comprises the
amino acid sequence of SEQ ID NO: 229 (humanized heavy chain sequence :
19
hVB22B p-z VH) , SEQ ID NO: 256 (humanized heavy chain sequence: hVB22B
g-e VH) , SEQ ID NO: 262 (humanized heavy chain sequence: hVB22B e
VH) , SEQ ID NO: 289 (humanized heavy chain sequence: hVB22B u2-wz4
VH) , or SEQ ID NO: 295 (humanized heavy chain sequence: hVB22B q-wz5
5 VH) ; or an antibody comprising a light chain variable region that
comprises the amino acid sequence of SEQ ID NO: 238 (humanized light
chain hVB22B p-z VL) , SEQ ID NO: 258 (humanized light chain hVB22B
g-e VL or hVB22B e VL) , SEQ ID NO: 291 (humanized light chain hVB22B
u2-wz4 VL) , or SEQ ID NO: 297 (humanized light chain hVB22B q-wz5
10 VL) . In particular, a preferred antibody is an antibody comprising
a heavy chain variable region and a light chain variable region of
any one of (1) to (5) indicated below:
(1) a heavy chain variable region comprising the amino acid sequence
of SEQ ID NO: 229, and a light chain variable region comprising the
15 amino acid sequence of SEQ ID NO: 238;
(2) a heavy chain variable region comprising the amino acid sequence
of SEQ ID NO: 256, and a light chain variable region comprising the
amino acid sequence of SEQ ID NO: 258;
(3) a heavy chain variable region comprising the amino acid sequence
20 of SEQ ID NO: 262, and a light chain variable region comprising the
amino acid sequence of SEQ ID NO: 258;
(4) a heavy chain variable region comprising the amino acid sequence
of SEQ ID NO: 289, and a light chain variable region comprising the
amino acid sequence of SEQ ID NO: 291; and
25 (5) a heavy chain variable region comprising the amino acid sequence
of SEQ ID NO: 295, and a light chain variable region comprising the
amino acid sequence of SEQ ID NO: 297.
Such antibodies include, for example, antibodies comprising the
amino acid sequence of SEQ ID NO: 2, 254 , 260, 287, or 293 (humanized
30 sc(Fv)2 sequence (hVB22B p-z sc(Fv)2, hVB22B g-e sc(Fv)2, hVB22B e
sc(Fv)2, hVB22B u2-wz4, or hVB22B q-wz5) .
The nucleotide sequence of hVB22B p-z VH is shown in SEQ ID NO:
228; the nucleotide sequence of hVB22B g-e VH is shown in SEQ ID NO:
255; the nucleotide sequence of hVB22B e VH is shown in SEQ ID NO:
35 261; the nucleotide sequence of hVB22B u2-wz4 VH is shown in SEQ ID
NO: 288; the nucleotide sequence of hVB22B q-wz5 VH is shown in SEQ
20
ID NO: 294; the nucleotide sequence of hVB22B p-z VL is shown in SEQ
ID NO: 237; the nucleotide sequences of hVB22B g-e VL and hVB22B e
VL are shown in SEQ ID NO: 257; the nucleotide sequence of hVB22B
u2-wz4 VL is shown in SEQ ID NO: 290; and the nucleotide sequence
of hVB22B q-wz5 VL is shown in SEQ ID NO: 296.
In the amino acid sequence of SEQ ID NO: 229 (humanized heavy
chain sequence: hVB22B p-z VH) , SEQ ID NO: 256 (humanized heavy chain
sequence: hVB22B g-e VH) , SEQ ID NO: 262 (humanized heavy chain
sequence: hVB22B e VH) , SEQ ID NO: 289 (humanized heavy chain sequence:
hVB22B u2-wz4 VH) , or SEQ ID NO: 295 (humanized heavy chain sequence:
hVB22B q-wz5 VH) ,
amino acids 31-35 correspond to CDR1;
amino acids 50-66 correspond to CDR2;
amino acids 99-107 correspond to CDR3;
amino acids 1-30 correspond to FR1;
amino acids 36-4 9 correspond to FR2;
amino acids 67-98 correspond to FR3; and
amino acids 108-118 correspond to FR4 .
In the amino acid sequence of SEQ ID NO: 238 (humanized light
chain sequence: hVB22B p-z VL) , SEQ ID NO: 258 (humanized light chain
sequence: hVB22B g-e VL or hVB22B e VL) , SEQ ID NO: 291 (humanized
light chain sequence: hVB22B u2-wz4 VL) , or SEQ ID NO: 297 (humanized
light chain sequence: hVB22B q-wz5 VL) ,
amino acids 24-39 correspond to CDRl;
amino acids 55-61 correspond to CDR2;
amino acids 94-102 correspond to CDR3;
amino acids 1-23 correspond to FR1;
amino acids 40-54 correspond to FR2 ;
amino acids 62-93 correspond to FR3; and
amino acids 103-112 correspond to FR4 .
In the present invention, SEQ ID NOs of the CDRs and FRs in the
hVB22B p-z VH sequence are shown below:
hVB22B p-z VH: FR1/SEQ ID NO: 230
hVB22B p-z VH: CDR1/SEQ ID NO: 36
hVB22B p-z VH: FR2/SEQ ID NO: 232
hVB22B p-z VH: CDR2/SEQ ID NO: 37
21
hVB22B p-z VH: FR3/SEQ ID NO: 234
hVB22B p-z VH: CDR3/SEQ ID NO: 38
hVB22B p-z VH: FR4/SEQ ID NO: 236.
In the present invention, SEQ ID NOs of the CDRs and FRs in the
5 hVB22B p-z VL sequence are shown below:
hVB22B p-z VL: FR1/SEQ ID NO: 239
hVB22B p-Z VL: CDR1/SEQ ID NO: 93
hVB22B p-z VL: FR2/SEQ ID NO: 241
hVB22B p-z VL: CDR2/SEQ ID NO: 94
10 hVB22B p-z VL: FR3/SEQ ID NO: 243
hVB22B p-z VL: CDR3/SEQ ID NO: 95
hVB22B p-z VL: FR4/SEQ ID NO: 245.
In the present invention, SEQ ID NOs of the CDRs and FRs in the
hVB22B g-e VH sequence are shown below:
15 hVB22B g-e VH: FR1/SEQ ID NO: 265
hVB22B g-e VH: CDR1/SEQ ID NO: 36
hVB22B g-e VH: FR2/SEQ ID NO: 267
hVB22B g-e VH: CDR2/SEQ ID NO: 37
hVB22B g-e VH: FR3/SEQ ID NO: 269
20 1YVB22B g-e VH: CDR3/SEQ ID NO: 38
hVB22B g-e VH: FR4/SEQ ID NO: 271.
In the present invention, SEQ ID NOs of the CDRs and FRs in the
1YVB22B g-e VL sequence are shown below:
hVB22B g-e VL: FR1/SEQ ID NO: 272
25 hVB22B g-e VL: CDR1/SEQ ID NO: 93
hVB22B g-e VL: FR2/SEQ ID NO: 274
hVB22B g-e VL: CDR2/SEQ ID NO: 94
hVB22B g-e VL: FR3/SEQ ID NO: 276
hVB22B g-e VL: CDR3/SEQ ID NO: 95
30 hVB22B g-e VL: FR4/SEQ ID NO: 278.
In the present invention, SEQ ID NOs of the CDRs and FRs in the
hVB22B e VH sequence are shown below:
hVB22B e VH: FR1/SEQ ID NO: 279
hVB22B e VH : CDRl/SEQ ID NO:' 36
35 hVB22B e VH: FR2/SEQ ID NO: 281
hVB22B e VH : CDR2/SEQ ID NO: 37
22
hVB22B e VH: FR3/SEQ ID NO: 283
hVB22B e VH: CDR3/SEQ ID NO: 38
hVB22B e VH: FR4/SEQ ID NO: 285.
In the present invention, SEQ ID NOs of the CDRs and FRs in the
5 hVB22B e VL sequence are shown below:
hVB22B e VL: FR1/SEQ ID NO: 272
hVB22B e VL : CDR1/SEQ ID NO: 93
hVB22B e VL: FR2/SEQ ID NO: 274
hVB22B e VL: CDR2/SEQ ID NO: 94
10 hVB22B e VL: FR3/SEQ ID NO: 276
hVB22B e VL: CDR3/SEQ ID NO: 95.
hVB22B e VL: FR4/SEQ ID NO: 278.
In the present invention, SEQ ID NOs of the CDRs and FRs in the
hVB22B u2-wz4 VH sequence are shown below:
15 hVB22B u2-wz4 VH : FR1/SEQ ID NO: 298
hVB22B u2-wz4 VH: CDR1/SEQ ID NO: 36
hVB22B u2-wz4 VH: FR2/SEQ ID NO: 299
hVB22B u2-wz4 VH: CDR2/SEQ ID NO: 37
hVB22B u2-wz4 VH: FR3/SEQ ID NO: 300
20 hVB22B u2-wz4 VH: CDR3/SEQ ID NO: 38
hVB22B u2-wz4 VH : FR4/SEQ ID NO: 301.
In the present invention, SEQ ID NOs of the CDRs and FRs in the
hVB22B
u2
-wz 4
VL
sequence are shown b
hVB22B
u2
-wz4
VL:
FR1/SEQ ID NO:
302
25
hVB22B
u2
-wz4
VL:
CDR1/SEQ ID NO:
93
hVB22B
u2
-wz4
VL:
FR2/SEQ ID NO:
303
hVB22B
u2
-wz4
VL:
CDR2/SEQ ID NO:
94
hVB22B
u2
-wz4
VL:
FR3/SEQ ID NO:
304
hVB22B
u2
-wz4
VL:
CDR3/SEQ ID NO:
95
30
hVB22B
u2
-wz4
VL:
FR4/SEQ ID NO:
305.
In the present invention, SEQ ID NOs of the CDRs and FRs in the
hVB22B q-wz5 VH sequence are shown below:
hVB22B q-wz5 VH : FRl/SEQ ID NO: 298
hVB22B q-wz5 VH : CDR1/SEQ ID NO: 36
35 hVB22B q-wz5 VH : FR2/SEQ ID NO: 299
hVB22B q-wz5 VH : CDR2/SEQ ID NO: 37
23
hVB22B q-wz5 VH : FR3/SEQ ID NO: 306
hVB22B q-wz5 VH : CDR3/SEQ ID NO: 38
hVB22B q-wz5 VH: FR4/SEQ ID NO: 301.
In the present invention, SEQ ID NOs of the CDRs and FRs in the
hVB22B q-wz5 VL sequence are shown below:
hVB22B q-wz5 VL: FRl/SEQ ID NO: 302
hVB22B q-wz5 VL: CDR1/SEQ ID NO: 93
hVB22B q-wz5 VL: FR2/SEQ ID NO: 307
hVB22B q-wz5 VL: CDR2/SEQ ID NO: 94
hVB22B q-wz5 VL: FR3/SEQ ID NO: 308
hVB22B q-wz5 VL: CDR3/SEQ ID NO: 95
hVB22B q-wz5 VL: FR4/SEQ ID NO: 305'.
The CDRs and FRs in the hVB22B p-z sequence, hVB22B g-e sequence,
hVB22B e sequence, hVB22B u2-wz4 sequence, and hVB22B q-wz5 sequence
are shown in Fig. 18.
In other embodiments, preferred humanized antibodies of the
present invention include:
humanized antibodies comprising a heavy chain variable region which
has FRl, 2, 3, and 4 comprising amino acid sequences of any one of
(1) to (5) indicated below:
(1) SEQ ID NOs: 230, 232, 234, and 236 (hVB22B p-z: H chain FRl, 2,
3, and 4) ,
(2) SEQ ID NOs: 265, 267, 269, and 271 (hVB22B g-e: H chain FRl, 2,
3, and 4) ,
(3) SEQ ID NOs: 279, 281, 283, and 285 (hVB22B e: H chain FRl, 2,
3, and 4) ,
(4) SEQ ID NOs: 298, 299, 300, and 301 (hVB22B u2-wz4: H chain FRl,
2, 3, and 4) , and
(5) SEQ ID NOs : 298, 299, 306, and 301 (hVB22B q-wz5: H chain FRl,
2, 3, and 4) ;
humanized antibodies comprising a light chain variable region which
has FRl, 2, 3, and 4 comprising amino acid sequences of any one of
(1) to (4) listed below:
(1) SEQ ID NOs : 239, 241, 243, and 245 (hVB22B p-z: L chain FRl, 2,
3, and 4) ,
(2) SEQ ID NOs: 272, 274, 276, and 278 (hVB22B g-e or hVB22.B e: L
24
chain FR1, 2, 3 r and 4),
(3) SEQ ID NOs: 302, 303, 304, and 305 (hVB22B u2-we4 : L chain FR1,
2 , 3 , and 4 ) , and
(4) SEQ ID NOs: 302, 307, 308, and 305 (hVB22B q-wz5: L chain FR1,
5 2, 3, and 4);
humanized antibodies comprising a heavy chain variable region which
has CDRl, 2 and 3 comprising amino acid sequences according to the
SEQ ID NOs listed below:
SEQ ID NOs: 36, 37, and 38 (hVB22B p-z, hVB22B g-e, hVB22B e, hVB22B
10 u2-wz4, or hVB22B q~wz5: H chain CDRl, 2, and 3); and
humanized antibodies comprising a light chain variable region which
has CDRl, 2 and 3 comprising amino acid sequences according to the
SEQ ID NOs listed below:
SEQ ID NOs: 93, 94, and 95 (hVB22B p-z hVB22B g-e, hVB22B e, hVB22B
15 u2-wz4, or hVB22B q-wz5: L chain CDRl, 2, and 3).
In yet another preferred embodiment, preferred humanized
antibodies of the present invention include:
humanized antibodies comprising heavy chain and light chain variable
regions of any one of (1) to (5) indicated below.
20 (1) a heavy chain variable region which comprises FR1, 2, 3, and 4
comprising the amino acid sequences of SEQ ID NOs: 230, 232, 234,
and 236, respectively, and a light chain variable region which
comprises FR1, 2, 3, and 4 comprising the amino acid sequences of
SEQ ID NOs: 239, 241, 243, and 245, respectively;
25 (2) a heavy chain variable region which comprises FR1 , 2, 3, and 4
comprising the amino acid sequences of SEQ ID NOs: 265, 267, 269,
and 271, respectively, and a light chain variable region which
comprises FR1, 2, 3, and 4 comprising the amino acid sequences of-
SEQ ID NOs: 272, 274, 276, and 278, respectively;
30 (3) a heavy chain variable region which comprises FR1, 2, 3 and 4
comprising the amino acid sequences of SEQ ID NOs: 279, 281, 283,
and 285, respectively, and a light chain variable region which
comprises FR1, 2, 3, and 4 comprising the amino acid sequences of
SEQ ID NOs: 272, 274, 276, and 278, respectively;
35 (4) a heavy chain variable region which comprises FR1, 2, 3, and 4
comprising the amino acid sequences of SEQ ID NOs: 298, 299, 300,
25
and 301, and a light chain variable region which comprises FR1, 2,
3, and 4 comprising the amino acid sequences of SEQ ID NOs: 302, 303,
304, and 305, respectively;
(5) a heavy chain variable region which comprises FR1, 2, 3, and 4
comprising the amino acid sequences of SEQ ID NOs: 298, 299, 306,
and 301, respectively, and a light chain variable region which
comprises FR1, 2, 3, and 4 comprising the amino acid, sequences of
SEQ ID NOs: 302, 307, 308, and 305, respectively; and
humanized antibodies comprising heavy chain and light chain variable
regions described below:
a heavy chain variable region which comprises CDR1, 2, and 3 comprising
the amino. acid sequences of SEQ ID NOs: 36, 37, and 38, respectively,
and a light chain variable region which comprises CDR1, 2, and 3
comprising the amino acid sequences of SEQ ID NOs: 93, 94, and 95,
respectively.
Chimeric antibodies and humanized antibodies exhibit lower
antigenicity in the human body, and thus are expected to be useful
when administered to humans for therapeutic purposes.
In one embodiment, the preferred antibodies of the present
invention include antibodies that bind to soluble Mpl . The term
"soluble Mpl" herein refers to Mpl molecules excluding those expressed
on the cell membrane. A specific example of a soluble Mpl is an Mpl
lacking the entire or a portion of the transmembrane domain. The
transmembrane domain of human Mpl corresponds to amino acids 492-513
in SEQ ID NO: 123.
An antibody that binds to soluble recombinant Mpl can be used
in detailed epitope analysis and kinetic analysis of receptor-ligand
binding, as well as for assessing the blood concentration and dynamic
behavior of the antibody in in vivo tests.
In one embodiment, the preferred antibodies of the present
invention include antibodies having binding activity against both
human and monkey Mpl . The present invention also provides antibodies
having agonistic activity to human Mpl and monkey Mpl. Antibodies
having agonistic activity to both human and monkey Mpl are expected
to be highly useful since the dynamic behavior and in vivo effects
of the antibody, which are generally difficult to determine in human
I
26
I
body, can be examined with monkeys.
Such antibodies may also have binding activity or agonistic
activity against Mpl from animals other than humans and monkeys (for
example, mice) .
5 In addition, the antibodies of the present invention include
antibodies with TPO agonistic activity (agonistic activity against
Mpl) of EC50 = 100 nM or lower, preferably EC50 =. 30 nM or lower,
more preferably EC50 = 10 nM or lower.
The agonistic activity can be determined by methods known to
10 those skilled in the art, for example, by the method described below.
The sequences for human Mpl (Palacios et al ., Cell 41: 727-734, (1985);
GenBank Accession NO. NM_005373) , cynomolgus monkey Mpl (the
nucleotide sequence and amino acid sequence are shown in SEQ ID NO:
1 64 and SEQ ID NO: 165, respectively), and mouse Mpl (GenBank Access ion
15 NO. NM_010823) are already known.
In addition, the present invention includes antibodies whose
binding activities to soluble Mpl are KD = 10" 6 M or lower, preferably
KD = 10" 7 M or lower.
In the present invention, whether the binding activity of an
20 antibody to soluble recombinant Mpl is KD = 10" 6 M or lower can be
determined by methods known to those skilled in the art. For example,
the activity can be determined using surface plasmon resonance with
Biacore. Specifically, soluble MPL-Fc protein , soluble MPL protein,
or epitope peptides recognized by antibodies are immobilized onto
25 sensor chips. Reaction rate constant can be determined by assessing
the interaction between the antibody and the soluble Mpl-Fc protein,
the soluble Mpl protein, or the epitope peptide recognized by the
antibody. The proteins to be immobilized on chips are not limited
in particular, and include, for example, MG10 (from Gln213 to
30 Ala231)-GST fusion protein and Mpl-IgG Fc fusion protein described
in the Examples. Since the antibodies are divalent and have two
antigen-binding sites, the binding activities of these antibodies
may be determined as those for the monovalent or divalent antibodies,
or for mixtures of both. Any of these can be used in the present
35 invention.
The binding activity can be evaluated by ELISA (enzyme-linked
27
immunosorbent assays), EIA (enzyme immunoassays), RIA (radio
immunoassays), or fluorescent antibody techniques. For example, in
enzyme immunoassays, a sample containing a test antibody, such as
purified antibody or culture supernatant of cells producing the test
5 antibody, is added to a plate coated with an antigen to which the
test antibody can bind. After incubating the plate with a secondary
antibody labeled with an enzyme such as alkaline phosphatase, the
plate is washed and an enzyme substrate such as p-nitrophenyl
phosphate is added. The antigen-binding activity can then be
10 evaluated by determining the absorbance.
There is no specific limitation as to the upper limit of the
binding activity; for example, the upper limit may be set within a
technically feasible range by those skilled in the art. However, the
technically feasible range may expand with the advancement of
15 technology.
In an embodiment, the preferred antibodies of the present
invention include antibodies recognizing epitopes that are recognized
by any one of the antibodies indicated in (I) to (XII) below. The
antibody of any one of (I) to (XII) is preferably a minibody.
20
(1) Antibody comprising a VH that has CDR1, 2, and 3 comprising the
amino acid sequences according to SEQ ID NOs in any one of (1) to
(17) indicated below (name of each antibody and the H chain CDR
contained in the antibody are indicated inside the parentheses) :
25 (1) SEQ ID NOs: 3, 4, and 5 (VA7 : H chain CDRl, 2, and 3),
(2) SEQ ID NOs: 6, 7, and 8 (VA130 or VB17B: H chain CDRl, 2, and
3) ,
(3) SEQ ID NOs: 9, 10, and 11 (VA259: H chain CDRl, 2, and 3),
(4) SEQ ID NOs: 15, 16, and 17 (VB12B: H chain CDRl, 2, and 3),
30 (5) SEQ ID NOs: 18, 19, and 20 (VB140: H chain CDRl, 2, and 3),
(6) SEQ ID NOs: 21, 22, and 23 (VB33: H chain CDRl, 2, and 3),
(7) SEQ ID NOs: 24, 25, and 26 (VB45B: H chain CDRl, 2, and 3),
(8) SEQ ID NOs: 27, 28, and 29 (VB8B : H chain CDRl, 2, and 3),
(9) SEQ ID NOs: 30, 31, and 32 (VB115: H chain CDRl, 2, and 3),
35 (10) SEQ ID NOs: 33, 34, and 35 (VB14B: H chain CDRl, 2, and 3),
(11) SEQ ID NOs: 36, 37, and 38 (VB22B, VB4B, hVB22B p-Z, hVB22B g-e,
28
hVB22B e, hVB22B u2-wz4 or hVB22B q-wz5: H chain CDRl, 2, and 3),
(12) SEQ ID NOs: 39, 40, and 41 (VB16: H chain CDRl, 2, and 3),
(13) SEQ ID NOs: 42, 43, and 44 (VB157: H chain CDRl, 2, and 3),
(14) SEQ ID NOs: 48, 49, and 50 (VB51: H chain CDRl, 2, and 3),
(15) SEQ ID NOs: 51, 52, and 53 (AB317: H chain CDRl, 2, and 3),
(16) SEQ ID NOs: 54, 55, and 56 (AB324: H chain CDRl, 2, and 3),
(17) SEQ ID NOs: 57, 58, and 59 (TA136: H chain CDRl, 2, and 3).
(II) Antibody comprising a VL which has CDRl, 2, and 3 comprising
the amino acid sequences according to SEQ ID NOs in any one of (1)
to (10) indicated below (name of each antibody and the L chain CDR
in the antibody are indicated inside the parentheses) :
(1) SEQ ID NOs: 60, 61, and 62 (VA7: L chain CDRl, 2, and 3),
(2) SEQ ID NOs: 63, 64, and 65 (VA130, VA259, VB17B, VB12B, VB140,
VB45B, VB115, VB14B or VB51: L chain CDRl, 2, and 3),
(3) SEQ ID NOs: 78, 79, and 80 (VB33 or VB157: L chain CDRl, 2, and
3) ,
(4) SEQ ID NOs: 84, 85, and 86 (VB8B: L chain CDRl, 2, and 3),
(5) SEQ ID NOs: 93, 94, and 95 (VB22B, hVB22B p-z, hVB22B g-e, hVB22B
e, hVB22B u2-wz4 or hVB22B q-wz5: L chain CDRl, 2, and 3),
(6) SEQ ID NOs: 96, 97, and 98 (VB16: L chain CDRl, 2, and 3),
(7) SEQ ID NOs: 102, 103, and 104 (VB4B: L chain CDRl, 2, and 3),
(8) SEQ ID NOs: 108, 109, and 110 (AB317: L chain CDRl, 2, and 3),
(9) SEQ ID NOs: 111, 112, and 113 (AB324: L chain CDRl, 2, and 3),
(10) SEQ ID NOs: 114, 115, and 116 (TA136: L chain CDRl, 2, and 3) .
(Ill) Antibody comprising a VH that comprises an amino acid sequence
of
the SEQ
ID
NO in
any one of
(1)
SEQ
ID
NO:
124
(VA7 : VH) ,
(2)
SEQ
ID
NO:
126
(VA130: VH).,
(3)
SEQ
ID
NO:
128.
(VA259: VH) ,
(4)
SEQ
ID
NO:
130
(VB17B: VH) ,
(5)
SEQ
ID
NO:
132
(VB12B: VH) ,
(6)
SEQ
ID
NO:
134
(VB140: VH) ,
(7)
SEQ
ID
NO:
136
(VB33: VH),
(8)
SEQ
ID
NO:
138
(VB45B: VH) ,
29
(9) SEQ ID NO: 140 (VB8B: VH) ,
(10)
SEQ
ID
NO:
142
(VB115: VH) ,
(11)
SEQ
ID
NO:
144
(VB14B: VH),
(12)
SEQ
ID
NO:
118
(VB22B: VH) ,
( 13 )
SEQ
ID
NO:
146
(VB16: VH) ,
SEO
ID
NO:
148
(VB157 : VH) ,
( lo )
X *J V
fVB4B- VH) ,
(16)
SEQ
ID
NO:
152
(VB51: VH),
(11)
SEQ
ID
NO:
155
(AB317: VH) ,
(18)
SEQ
ID
NO:
159
(AB324: VH) ,
(19)
SEQ
ID
NO:
162
(TA136: VH) ,
(20)
SEQ
ID
NO:
229
(hVB22B p-z: VH) ,
(21)
SEQ
ID
NO:
256
(hVB22B g-e: VH) ,
(22)
SEQ
ID
NO:
262
(hVB22B e: VH) ,
(23)
SEQ
ID
NO:
289
(hVB22B u2-wz4: VH) ,
(24)
SEQ
ID
NO:
295
(hVB22B q-wz5: VH) .
(IV) Antibody comprising a VL that comprises an amino acid sequence
of the SEQ ID NO in any one of (1) to (18):
(1) SEQ ID NO: 125 (VA7 : VL) ,
(2) SEQ ID NO: 127 (VA130, VB17B, VB12B, VB115 or VB14B: VL) ,
(3) SEQ ID NO: 129 (VA259: VL) ,
(4) SEQ ID NO: 135 (VB140 or VB45B: VL) ,
(5) SEQ ID NO: 137 (VB33: VL) ,
(6) SEQ ID NO: 141 (VB8B: VL) ,
(7) SEQ ID NO: 120 ( VB22B : VL) ,
(8) SEQ ID NO: 147 (VB16: VL) ,
(9) SEQ ID NO: 149 (VB157: VL) ,
(10) SEQ ID NO: 151 (VB4B: VL) ,
(11) SEQ ID NO: 153 (VB51: VL) ,
(12) SEQ ID NO: 157 (AB317: VL) ,
(13) SEQ ID NO: 161 (AB324: VL) ,
(14) SEQ ID NO: 163 (TA136: VL) ,
(15) SEQ ID NO: 238 (hVB22B p-z: VL) ,
(16) SEQ ID NO: 258 (hVB22B g-e: VL or hVB22B e: VL) ,
(17) SEQ ID NO: 291 (hVB22B u2-wz4: VL) ,
30
(18) SEQ ID NO: 297 (hVB22B q-wz5: VL) .
(V) Antibody comprising a VH and VL according to any one of (1) to
(18) :
5 (1) SEQ ID NOs: 3, 4, and 5 (VA7 : H chain CDR1, 2, and 3); SEQ ID
NOs: 60, 61, and 62 (VA7: L chain CDR1, 2, and 3),
(2) SEQ ID NOs: 6, 7, and 8 (VA130 or VB17B: H chain CDRl, 2, and
3), SEQ ID NOs: 63, 64, and 65 (VA130 or VB17B: L chain CDR1, 2, and
3) ,
10 (3) SEQ ID NOs: 9, 10, and 11 (VA259: H chain CDRl, 2, and 3); SEQ
ID NOs: 66, 67, and 68 (VA259: L chain CDRl, 2, and 3),
(4) SEQ ID NOs : 15, 16, and 17 ( VB12B : H chain CDRl, 2, and 3); SEQ
ID NO: 72, 73, and 74 (VB12B: L chain CDRl, 2, and 3),
(5) SEQ ID NOs: 18, 19, and 20 (VB140: H chain CDRl, 2, and 3); SEQ
15 ID NOs: 75, 76, and 77 (VB140: L chain CDRl, 2, and 3),
(6) SEQ ID NOs: 21, 22, and 23 (VB33: H chain CDRl, 2, and 3); SEQ
ID NOs: 78, 79, and 80 (VB33: L chain CDRl, 2, and 3),
(7) SEQ ID NOs : 24, 25, and 26 (VB45B: H chain CDRl, 2, and 3); SEQ
ID NOs: 81, 82, and 83 (VB45B: L chain CDRl, 2, and 3),
20 (8) SEQ ID NOs : 27, 23, and 29 (VB8B: H chain CDRl, 2, and 3); SEQ
ID NOs: 84-, 85, and 86 { VB8B: L chain CDRl, 2, and 3),
(9) SEQ ID NOs : 30, 31, and 32 (VB115: H chain CDRl, 2, and 3); SEQ
ID NOs: 87, 88, and 89 (VB115: L chain CDRl, 2, and 3),
(10) SEQ ID NOs: 33, 34, and 35 (VB14B: H chain CDRl, 2, and 3); SEQ
25 ID NOs: 90, 91, and 92 (VB14B: L chain CDRl, 2, and 3),
(11) SEQIDNOs: 36, 37, and 38 (VB22B, hVB22Bp-z, hVB22B g-e, hVB22B
e, hVB22B u2-wz4 or hVB22B q-wz5: H chain CDRl, 2, and 3); SEQ ID
NOs: 93, 94, and 95 (VB22B, hVB22B p-z, hVB22B g-e, hVB22B e, hVB22B
u2-wz4 or hVB22B q-wz5: L chain CDRl, 2, and 3),
30 (12) SEQ ID NOs: 39, 40, and 41 (VB16: H chain CDRl, 2, and 3); SEQ
ID NOs: 96, 97, and 98 (VB16: L chain CDRl, 2, and 3),
(13) SEQ ID NOs: 42, 43, and 44 (VB157: H chain CDRl, 2, and 3); SEQ
ID NOs: 99, 100, and 101 (VB157: L chain CDRl, 2, and 3),
(14) SEQ ID NOs: 45, 46, and 47 (VB4B: H chain CDRl, 2, and 3); SEQ
35 ID NOs: 102, 103, and 104 (VB4B: L chain CDRl, 2, and 3),
(15) SEQ ID NOs: 48, 49, and 50 (VB51: H chain CDRl, 2, and 3); SEQ
31
ID NOs: 105, 106, and 107 (VB51: L chain CDR1, 2, and 3),
(16) SEQ ID NOs r 51, 52, and 53 (AB317: H chain CDR1, 2, and 3); SEQ
ID NOs : 108, 109, and 110 (AB317: L chain CDR1, 2, and 3),
(17) SEQ ID NOs: 54, 55, and 56 (AB324: H chain CDR1, 2, and 3); SEQ
ID NOs : 111, 112, and 113 (AB324 : L chain CDR1, 2, and 3),
(18) SEQ ID NOs: 57, 58, and 59 (TA136: H chain CDR1 , 2, and 3); SEQ
ID NOs : 114, 115, and 116 (TA136: L chain CDR1, 2, and 3).
(VI) Antibody comprising a VH and a VL that comprise the amino acid
sequences according to SEQ ID NOs in any one of (1) to (24) indicated
below:
(1) SEQ ID NO: 124 (VA7 : VH) , SEQ ID NO: 125 (VA7 : VL) ,
(2) SEQ ID NO: 126 (VA130: VH) , SEQ ID NO: 127 (VA130: VL) ,
(3) SEQ ID NO: 128 (VA259: VH) , SEQ ID NO: 129 (VA259: VL) ,
(4) SEQ ID NO: 130 (VB17B: VH) , SEQ ID NO: 127 (VB17B: VL) ,
(5) SEQ ID NO: 132 (VB12B: VH ) , SEQ ID NO: 127 (VB12B: VL) ,
(6) SEQ ID NO: 134 (VB140: VH) , SEQ ID NO: 135 (VB140: VL) ,
(7) SEQ ID NO: 136 (VB33: VH) , SEQ ID NO: 137 (VB33: VL),
(8) SEQ ID NO: 138 (VB45B: VH) , SEQ ID NO: 135 (VB45B: VL) ,
(9) SEQ ID NO: 140 (VB8B: VH) , SEQ ID NO: 141 (VB8B: VL) ,
(10)
SEQ
ID
NO:
142
(VB115:
VH) ,
SEQ ID NO
127
(VB115: VL),
(11)
SEQ
ID
NO:
144
(VB14B:
VH) ,
SEQ ID NO
: 127
(VB14B: VL) ,
(12)
SEQ
ID
NO:
118
(VB22B :
VH) ,
SEQ ID NO
: 12 0
(VB22B: VL),
(13)
SEQ
ID
NO:
146
(VB16: VH) ,
SEQ ID NO:
147
(VB16: VL) ,
(14)
SEQ
ID
NO:
148
(VB157:
VH) ,
SEQ ID NO
: 149
(VB157: VL),
(15)
SEQ
ID
NO:
150
(VB4B: VH) ,
SEQ ID NO:
151
(VB4B: VL),
<16)
SEQ
ID
NO:
152
(VB51: VH) ,
SEQ ID NO:
153
(VB51: VL),
(17)
SEQ
ID
NO:
155
(AB317 :
VH) ,
SEQ ID NO
: 157
(AB317: VL),
(18)
SEQ
ID
NO:
159
(AB324 :
VH) ,
SEQ ID NO
: 161
(AB324: VL) ,
(19)
SEQ
ID
NO:
162
(TA136:
VH) ,
SEQ ID NO
: 163
(TA136: VL) ,
(20)
SEQ
ID
NO:
229
(hVB22B
p-z:
VH), SEQ
ID NO
: 238 (hVB22B
VL) ,
(21)
SEQ
ID
NO:
256
(hVB22B
g-e:
VH) , SEQ
ID NO
: 258 (hVB22B
VL) ,
(22)
SEQ
ID
NO:
262
(hVB22B
e: VH) , SEQ ID
NO: 258 (hVB22B e:
(23)
SEQ ID NO: 289 (hVB22B u2
-wz4
. VH) , SEQ ID NO:
291 (hVB22B u2
32
VL) ,
(24) SEQ ID NO: 295 (hVB22B q-wz5 : VH) , SEQ ID NO: 297 (HVB22B q-wz5:
VL) .
5 (VII) Antibody comprising the amino acid sequence of SEQ ID NO: 122
(VB22B: scFv) .
(VIII) Humanized antibody comprising an amino acid sequence
according to any one of SEQ ID NO: 2 (hVB22B p-z: sc(Fv)2), SEQ ID
10 NO: 254 (hVB22B g-e: sc(Fv)2), SEQ ID NO: 260 (hVB22B e: sc(Fv)2),
SEQ ID NO: 287 (hVB22B u2-wz4 : sc(Fv)2),' and SEQ ID NO: 293 (hVB22B
q-wz5 : sc (Fv) 2 ) .
(IX) Antibody comprising a VH which has FR1, 2, 3, and 4 comprising
15 amino acid sequences according to SEQ ID NOs in any one of (1) to
(5) indicated below:
(1) SEQ ID NOs: 230, 232, 234, and 236 (hVB22B p-z: H chain FR1, 2,
3, and 4 ) ,
(2) SEQ ID NOs: 265, 267, 269, and 271 (hVB22B g-e: H chain FR1, 2,
20 3, and 4 ) ,
(3) SEQ ID NOs: 279, 281, 283, and 285 (hVB22B e: H chain FR1, 2,
3, and 4 ) ,
(4) SEQ ID NOs: 298, 299, 300, and 301 (hVB22B u2-wz4 : H chain FR1,
2, 3, and 4) ,
25 (5) SEQ ID NOs: 298, 299, 306, and 301 (hVB22B q-wz5: H chain FR1 ,
2, 3, and 4 ) .
(X) Antibody comprising a VL which has FR1, 2, 3 and 4 comprising
amino acid sequences according to SEQ ID NOs in any one of (1) to
30 (4) indicated below:
(1) SEQ ID NOs: 239, 241, 243, and 245 (hVB22B p-z: L chain FR1 , 2,
3, and 4 ) ,
(2) SEQ ID NOs: 272, 274, 276, and 278 (hVB22B g-e or hVB22B e: L
chain FR1 , 2, 3, and 4),
35 (3) SEQ ID NOs: 302, 303, 304, and 305 (hVB22B u2-wz4: L chain FR1,
2, 3, and 4) ,
33
(4) SEQ ID NOs: 302, 307, 308, and 305 (hVB22B q-wz5: L chain FR1,
2, 3, and 4) .
(XI) Antibody comprising VH and VL according to any one of (1) to
5 (5) indicated below:
(1) VH having FR1, 2, 3, and 4 comprising the amino acid sequences
of SEQ ID NOs: 230, 232, 234, and 236, respectively, and VL having
FR1, 2, 3, and 4 comprising the amino acid sequences of SEQ ID NOs:
239, 241, 243, and 245, respectively;
10 (2) VH having FR1, 2, 3, and 4 comprising the amino acid sequences
of SEQ ID NOs: 265, 267, 269, and 271, respectively, and VL having
FR1, 2, 3, and 4 comprising the amino acid sequences of SEQ ID NOs:
272, 274, 276, and 278, respectively;
(3) VH having FR1, 2, 3, and 4 comprising the amino acid sequences
15 of SEQ ID NOs: 279, 281, 283, and 265, respectively, and VL having
FR1, 2, 3, and 4 comprising the amino acid sequences of SEQ ID NOs:
272, 274, 276, and 278, respectively;
(4) VH having FR1, 2, 3, and 4 comprising the amino acid sequences
of SEQ ID NOs: 298, 299, 300, and 301, respectively, and VL having
20 FR1, 2, 3, and 4 comprising the amino acid sequences of SEQ ID NOs:
302, 303, 304, and 305, respectively;
(5) VH having FR1, 2, 3, and 4 comprising the amino acid sequences
of SEQ ID NOs: 298, 299, 306, and 301, respectively, and VL having
FR1, 2, 3, and 4 comprising the amino acid sequences of SEQ ID NOs:
25 302, 307, 308, and 305, respectively.
(XII) Antibody comprising the amino acid sequence of SEQ ID NO: 264
(VB22B: sc (Fv) 2) .
An antibody comprising an amino acid sequence of any one of (I)
30 to (XII) indicated above, in which one or more amino acids have been
substituted, deleted, added, and/or inserted, wherein the antibody
has activity equivalent to that of the antibody of any one of (I)
to (XII) .
Herein, the phrase "functionally equivalent" means that an
35 antibody of interest has a biological or biochemical activity
comparable to that of an antibody of the present invention. Such
34
activities include, for example, binding activities and agonistic
activities .
Methods for preparing polypeptides functionally equivalent to
a certain polypeptide are well known to those skilled in the art,
5 and include methods of introducing mutations into polypeptides. For
example, those skilled in the art'can prepare an antibody functionally
equivalent to the antibodies of the present invention by introducing
appropriate mutations into the antibody using site-directed
mutagenesis (Hashimoto-Gotoh, T. et al. Gene 152, 271-275, (1995);
10 Zoller, MJ, and Smith, M. Methods Enzymol. 100, 468-500, (1983);
Kramer, W. et al., Nucleic Acids Res . 12, 9441-9456, (1984); Kramer,
W. and Fritz HJ, Methods Enzymol. 154, 350-367, (1987); Kunkel, TA,
Proc. Natl. Acad. Sci . USA. 82, 488-492, (1985); Kunkel, Methods
Enzymol. 85, 2763-2766, (1988)), or such . Amino acid mutations may
15 occur naturally. Thus, the present invention also comprises
antibodies functionally equivalent to the antibodies of the present
invention and comprising the amino acid sequences of these antibodies,
in which one or more amino acids is mutated. In such mutants, the
number of amino acids that may be mutated is not particularly
20 restricted, so long as the activity is maintained. Generally, the
number of amino acids that are mutated is 50 amino acids or less,
preferably 30 or less, more preferably 10 or less (for example, five
amino acids or less) . Likewise, the site of mutation is not
particularly restricted, so long as the mutation does not result in
25 the disruption of activity.
Amino acid mutations may be made at one or more predicted,
preferably nonessential, amino acid residues. A "nonessential"
amino acid residue is a residue that can be altered from the wild-type
sequence of a protein without altering the biological activity,
30 whereas an "essential" amino acid residue is required for biological
activity. An amino acid is preferably substituted for a different
amino acid(s) that allows the properties of the amino acid side-chain
to be conserved. Accordingly, throughout the present application,
a "conservative amino acid substitution" means a replacement of an
35 amino acid residue belonging to one of the following groups with
another amino acid in the same group having a chemically similar side
35
chain. Groups of amino acid residues having similar side chains have
been defined in the art. Examples of amino acid side chain properties
are: hydrophobic amino acids (A, I, L, M, F, P, W, Y, and V) , hydrophilic
amino acids ( R, D, N, C, E, Q, G, H, K, S, and T) , amino acids comprising
5 the following side chains: aliphatic side chains (G, A, V, L, I, and
P) ; hydroxyl-containing side chains (S, T, and Y) ; sulfur-containing
side chains (C and M) ; carboxylic acid- and amide-containing side
chains (D, N, E, and Q) ; basic side chains (R, K, and H) ; aromatic
ring-containing side chains (H, F, Y , and W) (amino acids are
10 represented by one-letter codes in parentheses) .
A polypeptide comprising a modified amino acid sequence, in
which one or more amino acid residues is deleted, added, and/or
replaced with other amino acids, is known to retain its original
biological activity (Mark, D. F. et al., Proc. Natl. Acad. Sci. USA
15 81, 5662-5666 (1984); Zoller, M. J. & Smith, M. Nucleic Acids Research
10, 6487-6500 (1982); Wang, A. et al., Science 224, 1431-1433;
Dalbadie-McFarland, G. et al., Proc. Natl. Acad. Sci. USA "79,
6409-6413 (1982) ) .
Fusion proteins containing antibodies that comprise the amino
20 acid sequence of an antibody of the present invention, in which two
or more amino acid residues have been added, are included in the
present invention. The fusion protein results from a fusion between
one of the above antibodies and a second peptide or protein, and is
included in the present invention. The fusion protein can be prepared
25 by ligating a polynucleotide encoding an antibody of the present
invention and a polynucleotide encoding a second peptide or
polypeptide in frame, inserting this into an expression vector, and
expressing the fusion construct in a host. Some techniques known to
those skilled in the art are available for this purpose. The partner
30 peptide or polypeptide to be fused with an antibody of the present
invention may be a known peptide, for example, FLAG (Hopp, T. P. et
al., BioTechnology 6, 1204-1210 (1988)), 6x His consisting of six
His (histidine) residues, lOx His, influenza hemagglutinin (HA) ,
human c-myc fragment, VSV-GP fragment, pl8HIV fragment, T7-tag,
35 HSV-tag, E-tag, SV40 T antigen fragment, lck tag, a-tubulin fragment,
B-tag, Protein C fragment. Other partner polypeptides to be fused
36
with the antibodies of the present invention include, for example,
GST (glutathione-S-transferase) , HA (influenza hemagglutinin),
immunoglobulin constant region, p-galactosidase, and MBP
(maltose-binding protein) . A polynucleotide encoding one of these
5 commercially available peptides or polypeptides can be fused with
a polynucleotide encoding an antibody of the present invention. The
fusion polypeptide can be prepared by expressing the fusion construct .
As described below, the antibodies of the present invention may
differ in amino acid sequence, molecular weight, isoelectric point,
10 presence/absence of sugar chains, and conformation depending on the
cell or host producing the antibody, or purification method . However,
a resulting antibody is included in the present invention, as long
as it is functionally equivalent to an antibody of the present
invention. For example, when an antibody of the present invention
15 is expressed in prokaryotic cells, for example E. coli, a methionine
residue is added to the N terminus of the original antibody amino
acid sequence. Such antibodies are included in the present
invention .
An antibody that recognizes an epitope recognized by the
20 antibody according to any one of (I) to (XII) indicated above is
expected to have a high agonistic activity. Such antibodies can be
prepared by methods known to those skilled in the art. The antibody
can be prepared by, for example, determining the epitope recognized
by the antibody according to any one of (I) to (XII) by conventional
25 methods, and using a polypeptide comprising one of the epitope amino
acid sequences as an immunogen. Alternatively, the antibody can be
prepared by determining the epitopes of conventionally prepared
antibodies and selecting an antibody that recognizes the epitope
recognized by an antibody of any one of (I) to (XII) .
30 In the present invention, a particularly preferred antibody is
an antibody that recognizes the epitope recognized by the antibody
comprising the amino acid sequence of SEQ ID NO: 2. The antibody
comprising the amino acid sequence of SEQ ID NO: 2 is predicted to
recognize the region from Glu 26 to Leu 274, preferably the region
35 from Ala 189 to Gly 245, more preferably the region from Gin 213 to
Ala 231 of human Mpl . Thus, antibodies recognizing the region of amino
37
acids 26 to 274, or amino acids 189 to 245, or amino acids 213 to
231 of human Mpl are also included in the present invention.
Antibodies recognizing regions of amino acids 26 to 274, amino
acids 189 to 245, or amino acids 213 to 231 of the human Mpl amino
5 acid sequence (SEQ ID NO: 123) can be obtained by methods known to
those skilled in the art. Such antibodies can be prepared by, for
example, using a peptide comprising amino acids 26 to 274, amino acids
189 to 245, or amino acids 213 to 231 of the human Mpl amino acid
sequence (SEQ ID NO: 123) as an immunogen. Alternatively, such
10 antibodies can be prepared by determining the epitope of a
conventionally prepared antibody and selecting an antibody that
recognizes the same epitope recognized by an antibody of the present
invention .
The present invention provides antibodies described above in
15 (I) to (XII) . In an embodiment of the present invention, a preferred
antibody is the one shown in (V) , a more preferred antibody is the
one shown in (VI), and a still more preferred is the one shown in
(VIII).
The present invention also provides vectors comprising
20 polynucleotides encoding the antibodies of the present invention,
or polynucleotides which hybridize under stringent conditions to the
polynucleotides of the present invention and encode antibodies having
activities equivalent to those of the antibodies of the present
invention. The polynucleotides of the present invention are polymers
25 comprising multiple bases or base pairs of deoxyribonucleic acids
(DNA) or ribonucleic acids (RNA) , and are not particularly limited,
as long as they encode the antibodies of the present invention. They
may also contain non-natural nucleotides . The polynucleotides of the
present invention can be used to express antibodies using genetic
30 engineering techniques. The polynucleotides of this invention can
also be used as probes in the screening of antibodies functionally
equivalent to the antibodies of the present invention. Specifically,
DNAs that hybridize under stringent conditions to a polynucleotide
encoding an antibody of the present invention, and encode antibodies
35 having activity equivalent to those of the antibodies of the present
invention can be obtained by techniques such as hybridization and
38
gene amplification (for example, PCR) , using a polynucleotide of the
present invention or a portion thereof as a probe. Such DNAs are also
included in the polynucleotides of the present invention.
Hybridization techniques are well known to those skilled in the art
5 (Sambrook, J et al . , Molecular Cloning 2nd ed. , 9.47-9.58, Cold Spring
Harbor Lab. press, 1989). Such hybridization conditions include, for
example, conditions of low stringency. Examples of conditions of low
stringency include post-hybridization washing in O.lx SSC and 0.1%
SDS at 42°C, and preferably in O.lx SSC and 0.1% SDS at 50°c. More
10 preferable hybridization conditions include those of high stringency.
Highly stringent conditions include, for example, washing in 5x SSC
and 0.1% SDS at 65 °C . In these conditions , the higher the temperature ,
the higher the expectation of efficiently obtaining polynucleotides
with a high homology. However, several factors, such as temperature
15 and salt concentration, can influence hybridization stringency, and
those skilled in the art can suitably select these factors to
accomplish similar stringencies.
Antibodies that are encoded by polynucleotides obtained by the
hybridization and gene amplification techniques, and are functionally
20 equivalent to the antibodies of the present invention generally
exhibit high homology to the antibodies of the this invention at the
amino acid level. The antibodies of the present invention include
antibodies that are functionally equivalent to the antibodies of the
present invention, and exhibit high amino acid sequence homology to
25 the antibodies of this invention. The term "high homology" generally
means identity at the amino acid level of at least 50% or higher,
preferably 75% or higher, more preferably 85% or higher, still more
preferably 95% or higher. Polypeptide homology can be determined by
the algorithm described in the report: Wilbur, W. J. and Lipman, D.
30 J. Proc. Natl. Acad. Sci. USA 80, 726-730 (1983).
When E. coli is used as a host, there is no particular limitation
as to the type of vector of the present invention, as long as the
vector contains an "ori" responsible for its replication in E. coli
and a marker gene. The "ori" ensures the amplification and mass
35 production of the vector in E. coli (for example, JM109, DH5a, HB101,
and XLlBlue) . The marker gene is used to select the E. coli
39
transf ormants (for example, a drug resistance gene selected by an
appropriate drug such as ampicillin r tetracycline, kanamycin, and
chloramphenicol) . The vectors include, for example, M13 vectors, pUC
vectors, pBR322, pBluescript, and pCR-Script . In addition to the
above vectors, for example, pGEM-T, pDIRECT, and pT7 can also be used
for the subcloning and excision of cDNAs.
In particular, expression vectors are useful as vectors of the
present invention. When an expression vector is expressed, for
example, in E. coli, it should have the above characteristics in order
to be amplified in E. coli. Additionally, when E . coli, such as JM109,
DH5a, HB101, or XLl-Blue are used as the host cell, the vector
preferably has a promoter, for example, lacZ, promoter (Ward et al.
(1989) Nature 341:544-546; (1992) FASEB J. 6:2422-2427), araB
promoter (Better et al . (1988) Science 240 : 1041-1043) , or T7 promoter ,
that allows efficient expression of the desired gene in E. coli. Other
examples of the vectors include pGEX-5X-l (Pharmacia), "QIAexpress
system" (QIAGEN) , pEGFP , and pET (where BL21, a strain expressing
T7 RNA polymerase, is preferably used as the host) .
Furthermore, the vectors may comprise a signal sequence for
polypeptide secretion. When producing polypeptides into the
periplasm of E . coli, the pelB signal sequence (Lei, S. P. et al.
J. Bacteriol. 169:4379 (1987)) may be used as a signal sequence for
polypeptide secretion. For example, calcium chloride methods or
electroporation methods may be used to introduce the vector into a
host cell.
In addition to E . coli, expression vectors derived from mammals
(e.g., P CDNA3 ( Invitrogen) , pEGF-BOS (Nucleic Acids Res. (1990)
18 (17) : 5322) , pEF, pCDM8), insect cells (e.g., "Bac-to-BAC
baculovirus expression system" (G1BCO-BRL) , pBacPAK8)., plants (e.g.,
pMHl, pMH2), animal viruses (e.g., pHSV, pMV, pAdexLcw) , retroviruses
(e.g., pZIPneo) , yeasts (e.g., "Pichia Expression Kit" (Invitrogen),
pNVll, SP-Q01) , and Bacillus subtilis (e.g., pPL608, pKTH50) may also
be used as a vector of the present invention.
In order to express proteins in animal cells such as CHO, COS,
and NIH3T3 cells, the vector preferably has a promoter necessary for
expression in such cells, for example, an SV40 promoter (Mulligan
40
et al. (1979) Nature 277:108), MMLV-LTR promoter, EFla promoter
(Mizushima et al. (1990) Nucleic Acids Res. 18:5322), CMV promoter,
etc.). It is even more preferable that the vector also carries a
marker gene for selecting transf ormants (for example, a
drug-resistance gene selected by a drug such as neomycin and G418.
Examples of vectors with such characteristics include pMAM, pDR2 ,
pBK-RSV, pBK-CMV, pOPRSV, and pOPl3, and such.
In addition, to stably express a gene and amplify the gene copy
number in cells, CHO cells that are defective in the nucleic acid
synthesis pathway are introduced with a vector containing a DHFR gene
(for example, pCHOI ) to compensate for the defect, and the copy number
is amplified using methotrexate (MTX) . Alternatively, a COS cell,
which carries an SV40 T antigen-expressing gene on its chromosome,
can be transformed with a vector containing the SV40 replication
origin (for example, pcD) for transient gene expression. The
replication origin may be derived from polyoma virus, adenovirus,
bovine papilloma virus (BPV) , and such. Furthermore, to increase the
gene copy number in host cells, the expression vector may contain,
as a selection marker, aminoglycoside transferase (APH) gene,
thymidine kinase (TK) gene, E. coli xanthine guanine phosphoribosyl
transferase (Ecogpt) gene, dihydrof olate reductase (dhfr) gene, and
such .
In the present invention, next, the vector is introduced into
a host cell. The host cells into which the vector is introduced are
not particularly limited, for example, E. coli and various animal
cells are available for this purpose. The host cells may be used,
for example, as a production system to produce and express the
antibodies of the present invention. In vitro and in vivo production
systems are available for polypeptide production systems.
Production systems that use eukaryotic cells or prokaryotic cells
are examples of in vitro production systems.
Eukaryotic cells that can be used are, for example, animal cells,
plant cells, and fungal cells. Known animal cells include: mammalian
cells, for example, CHO (J. Exp. Med. (1995)108, 945), COS, 3T3,
myeloma, BHK (baby hamster kidney), HeLa, Vero, amphibian cells such
as Xenopus laevis oocytes (Valle, etal. ( 1 981 ) Nature 2 91 , 358-340),
41
or insect cells (e.g., Sf9, Sf21, and Tn5) . In the present invention,
CHO-DG44, CHO-DXB11, C0S7 cells, and BHK cells can be suitably used.
Among animal cells, CHO cells are particularly favorable for
large-scale expression. Vectors can be introduced into a host cell
5 by, for example, calcium phosphate methods, the DEAE-dextran methods ,
methods using cationic liposome DOTAP (Boehringer-Mannheim) ,
electroporation methods, lipofection methods.
Plant cells include, for example, Nicotiana tabacum-derived
cells known as a protein production system. Calluses may be cultured
10 from these cells . Known fungal cells include yeast cells, for example,
genus Saccharomyces such as Saccharomyces cerevisiae and
Saccharomyces pombe; and filamentous fungi, for example, genus
Aspergillus such as Aspergillus niger.
Bacterial cells can be used in the prokaryotic production
15 systems. Examples of bacterial cells include E. coli (for example,
JM109, DH5a, HB101 and such); and Bacillus subtilis.
Next, the above host cells are cultured. Antibodies can be
obtained by transforming the cells with a polynucleotide of interest
and in vitro culturing of these transf ormants . Transf ormants can be
20 cultured using known methods . For example, DMEM, MEM, RPMI 1640, or
IMDM may be used as the culture medium for animal cells, and may be
used with or without serum supplements such as FBS or fetal calf serum
(FCS) . Serum-free cultures are also acceptable. The preferred pH
is about 6 to 8 during the course of culturing. Incubation is carried
25 out typically at a temperature of about 30 to 40°C for about 15 to
200 hours. Medium is exchanged, aerated, or agitated, as necessary.
On the other hand, production systems using animal or plant
hosts may be used as systems for producing polypeptides in vivo. For
example, a polynucleotide of interest is introduced into an animal
30 or plant and the polypeptide is produced in the body of the animal
or plant and then recovered. The "hosts" of the present invention
includes such animals and plants.
Animals to be used for the production system include mammals
or insects. Mammals such as goats, pigs, sheep, mice, and cattle may
35 be used (Vicki Glaser SPECTRUM Biotechnology Applications (1993)).
Alternatively, the mammals may be transgenic animals.
42
For example, a polynucleotide of interest is prepared as a
fusion gene with a gene encoding a polypeptide specifically produced
in milk, such as the goat P-casein gene. DNA fragments containing
the fusion gene are injected into goat embryos, which are then
5 introduced back to f emale goats . The desired antibody can be obtained
from milk produced by the transgenic goats, which are born from the
goats that received the embryos, or from their offspring.
Appropriate hormones may be administered to increase the volume of
milk containing the antibody produced by the transgenic goats (Ebert,
10 K.M. et al., Bio/Technology 12, 699-702 (1994)).
Insects, such as silkworms, may also be used. Baculoviruses
carrying a polynucleotide encoding an antibody of interest can be
used to infect silkworms, and the antibody of interest can be obtained
from the body fluids (Susumu, M. et al., Nature 315, 592-594 (1985)).
15 Plants used in the production system include, for example,
tobacco. When tobacco is used, a polynucleotide encoding an antibody
of interest is inserted into a plant expression vector, for example,
pMON 530, and then the vector is introduced into a bacterium, such
as Agrobacter ium tumef aciens . The bacteria are then used to infect
20 tobacco such as Nicotiana tabacum, and the desired antibodies can
be recovered from the leaves (Julian K. -C . Ma et al . , Eur . J. Immunol .
24, 131-138 (1994) ) .
The resulting antibody may be isolated from the inside or
outside (such as the medium) of host cells, and purified as a
25 substantially pure and homogenous antibody. Methods are not limited
to any specific method and any standard method for isolating and
purifying antibodies may be used. Polypeptides may be isolated and
purified, by selecting an appropriate combination of, for example,
chromatographic columns, filtration, ultrafiltration, salting out,
30 solvent precipitation, solvent extraction, distillation,
immunoprecipitation, SDS-polyacrylamide gel electrophoresis,
isoelectric focusing, dialysis, recrystallization, and others. The
term "substantially pure" as used herein in reference to a given
polypeptide means that the polypeptide is substantially free from
35 contaminants such as other biological macromolecules , culture media
(if recombinantly produced), or chemical precursors (if chemically
43
synthesized). The substantially pure polypeptide is at least 75%,
preferably at least about 80%, more preferably at least about 85,
90, 95, or 99% pure by dry weight. Purity can be measured by any
appropriate standard method, for example by a chromatography method,
5 polyacrylamide gel electrophoresis, or HPLC analysis.
Chromatographies include, for example, affinity
chromatographies, ion exchange chromatographies, hydrophobic
chromatographies, gel filtrations, reverse-phase chromatographies,
and adsorption chromatographies (Strategies for Protein Purification
10 and Characterization: A Laboratory Course Manual . Ed Daniel R. Marshal
et al., Cold Spring Harbor Laboratory Press, 1996). These
chromatographies can be carried out using liquid phase
chromatographies such as HPLC and FPLC. Examples of the affinity
chromatography columns include protein A columns and protein G columns .
15 Examples of the proteins A columns include Hyper D, POROS, and
Sepharose F. F. (Pharmacia) .
An antibody can be modified freely and peptide portions deleted
by treating the antibody with an appropriate protein modifying enzyme
before or after antibody purification. Such protein modifying
20 enzymes include, for example, trypsins, chymotrypsins, lysyl
endopeptidases, protein kinases, and glucosidases .
Antibodies that bind to Mpl can be prepared by methods known
to those skilled in the art.
For example, monoclonal antibody-producing hybridomas can be
25 essentially generated by known technologies as follows: immunizing
animals with Mpl proteins or Mpl-expressing cells as sensitized
antigens using conventional immunological methods; fusing the
obtained immunocytes with known parental cells by conventional cell
fusion methods; and screening for monoclonal antibody-producing cells
30 by conventional methods.
Specifically, monoclonal antibodies can be prepared by the
method below.
First, Mpl protein, which is used as a sensitized antigen for
preparing antibodies, is prepared by expressing the Mpl gene/amino
35 acid sequence (GenBank accession number: NM_005373). More
specifically, the gene sequence encoding Mpl is inserted into a known
44
expression vector, which is then transf ected into an appropriate host
cell. The subject human Mpl protein is purified from the host cell
or culture supernatant using known methods.
The purified Mpl protein is then used as a sensitized antigen.
Alternatively, a partial Mpl peptide may be used as a sensitized
antigen. In this case, the partial peptide can also be chemically
synthesized based on the amino acid sequence of human Mpl.
The epitopes of Mpl molecule that are recognized by an anti-Mpl
antibody of the present invention are not limited to a particular
epitope, and may be any epitope on the Mpl molecule. Thus, any
fragment can be used as an antigen for preparing anti-Mpl antibodies
of the present invention, as long as the fragment comprises an epitope
of the Mpl molecule.
There is no limitation as to the type of mammalian species to
be immunized with the sensitized antigen. However, a mammal is
preferably selected based on its compatibility with the parental cell
to be used in cell fusion. Generally, rodents (for example, mice,
rats, and hamsters), rabbits, and monkeys can be used.
Animals can be immunized with a sensitized antigen by known
methods such as a routine method of injecting a sensitized antigen
into a mammal intraper itoneally or subcutaneously . Specifically,
the sensitized antigen is diluted appropriately with
phosphate-buffered saline (PBS), physiological saline and such, and
then suspended. An adequate amount of a conventional adjuvant, for
example, Freund' s complete adjuvant, is mixed with the suspension,
as necessary. An emulsion is then prepared for administering to a
mammal several times over a 4- to 21-day interval. An appropriate
carrier may be used for the sensitized antigen in immunization.
A mammal is immunized as described above. After a titer
increase of target antibody in the serum is confirmed, immunocytes
are collected from the mammal and then subjected to cell fusion.
Spleen cells are the preferred immunocytes.
Mammalian myeloma cells are used as the parental cells to be
fused with the above immunocytes . Preferable myeloma cells to be used
include various known cell lines, for example, P3 ( P3x63Ag8 . 653 )
(Kearney JF, et al., J. Immnol. 123, 1548-1550 (1979)), P3x63Ag8U.l
45
(Yelton DE, et al., Current Topics in Microbiology and Immunology
81, 1-7 (1978)), NS-1 (Kohler, G. and Milstein, C. Eur. J. Immunol.
6, 511-519 (1976)), MPC-11 (Margulies, D. H. et al., Cell 8, 405-415
(1976)), SP2/0 (Shulman, M. et al., Nature 276, 269-270 (1978)), FO
5 (deSt. Groth, S. F. et al., J. Immunol. Methods 35, 1-21 (1980)),
S194 (Trowbridge, I. S., J. Exp. Med. 148, 313-323 (1978)), and R210
(Galfre, G. et al., Nature 277, 131-133 (1979)).
Cell fusions between the immunocytes and the myeloma cells as
described above can be essentially carried out using known methods,
10 for example, a method by Kohler and Milstein (Kohler, G. and Milstein,
C, Methods Enzymol. 73, 3-46 (1981)).
More specifically, the above-described cell fusions are carried
out, for example, in a conventional culture medium in the presence
of a cell fusion-promoting agent. The fusion-promoting agents
15 include, for example, polyethylene glycol (PEG) and Sendai virus (HVJ) .
If required, an auxiliary substance such as dimethyl sulfoxide may
also be added to improve fusion efficiency.
The ratio of immunocytes to myeloma cells may be determined at
one's own discretion, preferably, for. example, one myeloma cell for
20 every one to ten immunocytes. Culture media to be used for the above
cell fusions include, for example, media that are suitable for the
growth of the above myeloma cell lines, such as RPMI 1640 media and
MEM media, and other conventional culture media used for this type
of cell culture. In addition, serum supplements such as fetal calf
25 serum (FCS) may also be used in combination.
Cell fusion is carried out as follows. As described above,
predetermined amounts of immunocytes and myeloma cells are mixed well
in the culture medium. PEG solution (for example, mean molecular
weight of about 1, 000-6, 000) pre-heated to 37°C is added to the cell
30 suspension typically at a concentration of 30% to 60% (w/v) , and mixed
to produce fused cells (hybridomas) . Then, an appropriate culture
medium is successively added to the mixture, and the sample is
centrifuged to remove supernatant. This treatment is repeated
several times to remove the unwanted cell fusion-promoting agent and
35 others that are unfavorable to hybridoma growth.
Screening of the resulting hybridomas can be carried out by
46
culturing them in a conventional selective medium, for example,
hypoxanthine, aminopterin, and thymidine (HAT) medium. Culturing in
the above-descried HAT medium is continued for a period long enough
{typically, for several days to several weeks) to kill cells
5 (non-fused cells) other than the desired hybridomas. Then,
hybridomas are screened for single-cell clones capable of producing
the target antibody by conventional limiting dilution methods.
In addition to the method for preparing the above-descried
hybridomas by immunizing non-human animals with antigens, preferred
10 human antibodies having binding activity to Mpl can also be obtained
by: sensitizing human lymphocytes with Mpl in vitro; and fusing the
sensitized lymphocytes with human myeloma cells capable of dividing
permanently (see, Examined Published Japanese Patent Application No.
(JP-B) Hei 1-59878). Alternatively, it is possible to obtain human
15 antibodies against Mpl from immortalized cells producing anti-Mpl
antibodies. In this method, the cells producing anti-Mpl antibodies
are prepared by administering Mpl as an antigen to transgenic animals
comprising a repertoire of the entire human antibody genes (see, WO
94/25585, WO 93/12227, WO 92/03918, and WO 94/02602).
20 ■ The monoclonal antibody-producing hybridomas thus prepared can
be passaged in a conventional culture medium, and stored in liquid
nitrogen over long periods of time.
Monoclonal antibodies can be prepared from the above-described
hybridomas by, for example, a routine procedure of culturing the
25 hybridomas and obtaining antibodies from the culture supernatants .
Alternatively, monoclonal antibodies can be prepared by injecting
the hybridomas into a compatible mammal; growing these hybridomas
in the mammal; and obtaining antibodies from the mammal's ascites.
The former method is suitable for preparing highly purified antibodies,
30 while the latter is suitable for preparing antibodies on a large scale.
Recombinant antibodies can also be prepared by: cloning an
antibody gene from a hybridoma; inserting the gene into an appropriate
vector; introducing the vector into a host; and producing the
antibodies by using genetic recombination techniques (see, for
35 example, Vandamme, A. M. et al., Eur. J. Biochem. 192, 767-775,
(1990) ) .
47
Specifically, an mRNA encoding the variable (V) region of
anti-Mpl antibody is isolated from hybridomas producing the anti-Mpl
antibodies. For mRNA isolation, total RNAs are first prepared by
conventional methods such as guanidine ultracentrif ugation methods
(Chirgwin, J. M. et al., Biochemistry 18, 5294-5299 (1979)), or acid
guanidinium thiocyanate-phenol-chlorof orm (AGPC) methods
(Chomczynski, P. et al., Anal. Biochem. 162, 156-159 (1987)), and
then the target mRNA is prepared using an mRNA Purification Kit
(Pharmacia) and such. Alternatively, the mRNA can be directly
prepared using the QuickPrep mRNA Purification Kit (Pharmacia) .
A cDNA of the antibody V region is synthesized from the resulting
mRNA using reverse transcriptase. cDNA synthesis is carried out
using the AMV Reverse Transcriptase First-strand cDNA Synthesis Kit
(Seikagaku Co. ) , or such. Alternatively, cDNA can be synthesized and
amplified by the 5' -RACE method (Frohman, M. A. et al., Proc. Natl.
Acad. Sci. USA 85, 8998-9002 (1988); Belyavsky, A. et al., Nucleic
Acids Res. 17, 2919-2932 (1989)) using the 5'-Ampli FINDER RACE Kit
(Clontech) and PCR.
Target DNA fragments are purified from the obtained PCR products
and then ligated with vector DNAs to prepare recombinant vectors.
The vectors are introduced into E. coli and such, and colonies are
selected for preparing the recombinant vector of interest. The
target DNA nucleotide sequence is then confirmed by conventional
methods such as the dideoxynucleot ide chain termination method.
Once a DNA encoding the V region of target anti-Mpl antibody
is obtained, the DNA is inserted into an expression vector which
comprises a DNA encoding the constant region (C region) of a desired
antibody.
The method for producing anti-Mpl antibodies to be used in the
present invention typically comprises the steps of: inserting an
antibody gene into an expression vector, so that the gene is expressed
under the regulation of expression regulatory regions, such as
enhancer and promotor ; and transforming host cells with the resulting
vectors to express antibodies.
For expressing the antibody gene, polynucleotides encoding H
chain and L chain, respectively, are inserted into separate expression
48
vectors and co-transf ected into a host cell. Alternatively,
polynucleotides encoding both H chain and L chain are inserted into
a single expression vector and transfected into a host cell (see WO
94/11523) .
5 The term "agonistic activity" refers to an activity to induce
changes in some, biological activities through signal transduction
.into cells and such, due to the binding of an antibody to a receptor
antigen. The biological activities include, for example,
proliferation-promoting activities, proliferation activities,
10 viability activities, differentiation-inducing activities,
differentiation activities, transcriptional activities, membrane
transport activities, binding activities, proteolytic activities,
phosphorylation/dephosphorylation activities, oxidation/ reduction
activities, transfer activities, nucleolytic activities,
15 dehydration activities, cell death-inducing activities, and
apoptosis-inducing activities, but is not limited thereto.
The term "agonistic activity against Mpl" typically refers to
the activity of promoting the differentiation of megakaryocytes or
their parental hemopoietic stem cells into platelets, or the activity
20 of stimulating platelet proliferation.
Agonistic activity can be assayed by methods known to those
skilled in the art. The agonistic activity may be determined using
the original activity or a different activity as an indicator.
For example, agonistic activity can be determined by a method
25 using cell growth as an indicator as described in Examples. More
specifically, an ant ibody whose agonistic activity is to be determined
is added to cells which proliferate in an agonist-dependent manner,
followed by incubation of the cells. Then, a reagent such as WST-8
which shows a coloring reaction at specific wavelengths depending
30 on the viable cell count, is added to the culture and absorbance is
measured. The agonistic activity can be determined using the
measured absorbance as an indicator.
Cells that proliferate in an agonist-dependent manner can also
be prepared by methods known to those skilled in the art . For example,
35 when the antigen is a receptor capable of transducing cell growth
signals, cells expressing the receptor may be used. Alternatively,
49
when the antigen is a receptor that cannot transduce signals, a
chimeric receptor consisting of the intracellular domain of a receptor
that transduces cell growth signals and the extracellular domain of
a receptor that does not transduce cell growth signals can be prepared
for cellular expression. Receptors that transduce cell growth
signals include, for example, G-CSF receptors, mpl, neu, GM-CSF
receptors, EPO receptors, c-kit, and FLT-3. Cells that can be used
to express a receptor include, for example, BaF3, NFS 60, FDCP-1,
FDCP-2, CTLL-2, DA-1, and KT-3.
There is no limitation as to the type of detection indicators
to be used for determining agonistic activity, as long as the indicator
can monitor quantitative and/or qualitative changes. For example,
it is possible to use cell-free assay indicators, cell-based assay
indicators, tissue-based assay indicators, and in vivo assay
indicators. Indicators that can be used in cell-free assays include
enzymatic reactions, quantitative and/or qualitative changes in
proteins, DNAs, or RNAs. Such enzymatic reactions include, for
example, amino acid transfers, sugar transfers, dehydrations,
dehydrogenations, and substrate cleavages. Alternatively, protein
phosphorylations, dephosphorylations, dimerizations,
multimerizations, hydrolyses, dissociations and such; DNA or RNA
amplifications, cleavages, and extensions can be used as the indicator
in cell-free assays. For example, protein phosphorylations
downstream of a signal transduction pathway may be used as a detection
indicator. Alterations in cell phenotype, for example, quantitative
and/or qualitative alterations in products, alterations in growth
activity, alterations in cell number, morphological alterations, or
alterations in cellular properties, can be used as the indicator in
cell-based assays. The products include, for example, secretory
proteins, surface antigens, intracellular proteins, andmRNAs. The
morphological alterations include, for example, alterations in
dendrite formation and/or dendrite number, alteration in cell
flatness, alteration in cell elongation/axial ratio, alterations in
cell size, alterations in intracellular structure,
heterogeneity/homogeneity of cell populations, and alterations in
cell density. Such morphological alterations can be observed under
50
a microscope. Cellular properties to be used as the indicator include
anchor dependency, cytokine-dependent response, hormone dependency,
drug resistance, cell motility, cell migration activity, pulsatory
activity, and alteration in intracellular substances . Cell motility
5 includes cell infiltration activity and cell migration activity . The
alterations in intracellular substances include, for example,
alterations in enzyme activity, mRNA levels, levels of intracellular
signaling molecules such as Ca 2+ and cAMP, and intracellular protein
levels. When a cell membrane receptor is used, alterations in the
10 cell proliferating activity' induced by receptor stimulation can be
used as the indicator. The indicators to be used in tissue-based
assays include functional alterations adequate for the subject tissue.
In in vivo assays, alterations in tissue weight, alterations in the
blood system (for example, alterations in blood cell counts, protein
15 contents, or enzyme activities), alterations in electrolyte levels,
and alterations in the circulating system (for example, alterations
in blood pressure or heart rate) .
The methods for measuring such detection indices are not
particularly limited. For example, absorbance, luminescence, color
20 development, fluorescence, radioactivity, fluorescence polarization,
surface plasmon resonance signal, time-resolved fluorescence, mass,
absorption spectrum, light scattering, and fluorescence resonance
energy transfer may be used. These measurement methods are known to
those skilled in the art and may be selected appropriately depending
25 on the purpose. For example, absorption spectra can be obtained by
using a conventional photometer, plate reader, or such; luminescence
can be measured with a luminometer or such; and fluorescence can be
measured with a fluorometer or such. Mass can be determined with a
mass spectrometer. Radioactivity can be determined with a device
30 such as a gamma counter depending on the type of radiation.
Fluorescence polarization can be measured with BEACON (TaKaRa).
Surface plasmon resonance signals can be obtained with BIACORE.
Time-resolved fluorescence, fluorescence resonance energy transfer,
or such can be measured with ARVO or such. Furthermore, a flow
35 cytometer can also be used for measuring. It is possible to use one
of the above methods to measure two or more different types of
detection indices. A greater number of detection indices may also
be examined by using two or more measurement methods simultaneously
and/or consecutively. For example, fluorescence and fluorescence
resonance energy transfer can be measured at the same time with a
f luoronteter .
The present invention also provides pharmaceutical
compositions comprising antibodies of this invention. The
pharmaceutical compositions comprising antibodies of the present
invention are useful for treating and/or preventing thrombocytopenia
and such. Time required for the platelet count to recover to the
normal level can be shortened by administering an antibody of the
present invention after donation of platelet components. The amount
of platelet components at the time of blood collection can be increased
by pre-administering an antibody of the present invention.
When used as pharmaceutical compositions, the antibodies of the
present invention can be formulated by methods known to those skilled
in the art. For example, the antibodies can be administered
parenterally by injection of a sterile solution or suspension in water
or other pharmaceutically acceptable solvents. For example, the
antibodies can be formulated by appropriately combining with
pharmaceutically-acceptable carriers or solvents, specifically,
sterile water or physiological saline, vegetable oils, emulsifiers,
suspending agents, surfactants, stabilizers, flavoring agents,
excipients, vehicles, preservatives, binding agents, and such, and
mixing at a unit dosage and form required by accepted pharmaceutical
implementations. In such formulations, the amount of the thus
obtained active ingredient should be within the required range.
A sterile composition to be injected can be formulated using
a vehicle such as distilled water used for injection, according to
standard protocols.
Aqueous solutions used for injections include, for example,
physiological saline and isotonic solutions comprising glucose or
other adjunctive agents such as D-sorbitol, D-mannose, D-mannitol,
and sodium chloride. They may also be combined with an appropriate
solubilizing agent such as alcohol, specifically, ethanol,
polyalcohol such as propylene glycol or polyethylene glycol, or
52
non-ionic detergent such as polysorbate 80 or HCO-50, as necessary.
Oil solutions include sesame oils and soybean oils, and can be
combined with solubilizing agents such as benzyl benzoate or benzyl
alcohol. Injection solutions may also be formulated with buffers,
5 for example, phosphate buffers or sodium acetate buffers; analgesics,
for example, procaine hydrochloride; stabilizers, for example, benzyl
alcohol or phenol; or anti-oxidants . The prepared injections are
typically aliquoted into appropriate ampules.
The administration is preferably carried out parenterally ,
10 specifically, by injection, intranasal administration,
intrapulmonary administration, percutaneous administration, or such
Injections include, for example, intravenous injections,
intramuscular injections, intraperitoneal injections, and
subcutaneous injections. The injection solutions can be also
15 administered systemically or locally.
The administration methods can be selected properly according
to the patient's age, condition, and such. The applied dose of a
pharmaceutical composition comprising an antibody or polynucleotide
encoding the antibody may be, for example, in the range of 0.0001
20 to 1,000 mg/kg body weight. Alternatively, the dosage may be, for
example, in the range of 0 . 001 to 100, 000 mg/kg body weight . However,
the dosage is not restricted to the values described above. The dosage
and administration methods depend on the patient's weight, age, and
condition, and are appropriately selected by those skilled in the
25 art.
Furthermore, the present invention relates to methods for
inducing signals in Mpl-expressing cells by using the antibodies of
the present invention. More specifically, the present invention
relates methods for inducing signals in Mpl-expressing cells, in which
30 the methods comprise the step of contacting the cells with the
antibodies of the present invention.
All patents, published patent applications, and publications
cited herein are incorporated by reference in their entirety.
35 Examples
The present invention is specifically illustrated below with
53
reference to Examples, but it is not to be construed as being limited
thereto .
[Example 1] Preparation of anti-human Mpl antibodies
1.1 Establishment of Mpl-expressing BaF3 cell lines
BaF3 cell lines expressing the full-length Mpl gene were
established to obtain cell lines that proliferate in a TPO-dependent
manner .
A full-length human Mpl cDNA (Palacios, R. et al., cell, 41,
727-734 (1985)) (GenBank accession NO. NM_005373) was amplified by
PCR. The cDNA was cloned into a pC0S2 expression vector to construct
pCOS2-hMplf ull . The expression vector pC0S2 was constructed by
removing the DHFR gene expression region from pCHOI (Hirata, Y. et
al., FEBS Letter, 356, 244-248 (1994)), where the expression region
of the neomycin resistance gene HEF-VH-g^l (Sato, K. et al., Mol
Immunol., 31, 371-381 (1994)) is inserted.
The cynomolgus monkey Mpl cDNA (SEQ ID NO: 164) was cloned from
total RNA extracted from the bone marrow cells of cynomolgus monkey,
using a SMART RACE cDNA Amplification Kit (Clontech) . The resulting
cynomolgus monkey cDNA was inserted into pC0S2 to construct
pC0S2-monkeyMplf ull .
Then, the full-length mouse Mpl cDNA (GenBank accession NO.
NM_010823) was amplified by PCR, and inserted into pC0S2 to construct
pC0S2-mouseMplf ull.
Each vector (20 \xg) prepared as described above was mixed with
BaF3 cells (lx 10 7 cells/mL) suspended in PBS in Gene Pulser cuvettes.
This mixture was then pulsed at 0.33 kV and 950 fiFD using a Gene Pulser
II (Bio-Rad) . The BaF3 cells introduced with the above DNAs by
electroporation were added to RPMI 1640 medium (Invitrogen)
containing 1 ng/mL mouse interleukin 3 (hereinafter abbreviated as
mIL-3; Peprotech) , 500 ug/mL Geneticin (Invitrogen), and 10% FBS
(Invitrogen), and selected to establish a human Mpl-expressing BaF3
cell line (hereinafter abbreviated as "BaF3-human Mpl"), monkey
Mpl-expressing BaF3 cell line (hereinafter abbreviated as BaF3-monkey
Mpl) , and mouse Mpl-expressing BaF3 cell line (hereinafter
abbreviated as "BaF3-mouse Mpl") . Following selection, these cells
54
were cultured and maintained in RPMI 1640 containing 1 ng/mL rhTPO
(R&D) and 10% FBS.
1.2 Establishment of Mpl-expressing CHO cell lines
5 CHO cell lines expressing the full-length Mpl gene were
established to obtain cell lines to be used for assessing binding
activity by flow cytometry.
First, the DHFR gene expression site from pCHOI was inserted
into pCXN2 (Niwa, H. et al., Gene, 108, 193-199 (1991) ) at the Hindlll
10 site to prepare a pCXND3expression vector. The respective Mpl genes
were amplified by PCR using pC0S2-hMpl f ull, pC0S2-monkeyMplf ull, and
pCOS2-mouseMplfull as templates, and primers with a His-tag sequence .
The PCR products were cloned into pCXND3 to construct pCXND3-hMpl-His ,
pCXND3-monkey Mpl-His, and pCXND3-mouse Mpl-His, respectively.
15 Vectors thus prepared (25 ug each) were mixed with a PBS
suspension of CHO-DG44 cells (lx 10 1 cells/mL) in Gene Pulser cuvettes .
The mixture was then pulsed at 1.5 kV and 25 uTD using Gene Pulser
II (Bio-Rad) . The CHO cells introduced with these DNAs by
electroporation were added to CHO-S-SFMII medium (Invitrogen)
20 containing 500 ug/mL Geneticin and lx HT (Invitrogen). A human
Mpl-expressing CHO cell line (hereinafter abbreviated as "CHO-human
Mpl"), monkey Mpl-expressing CHO cell line (hereinafter abbreviated
as "CHO-monkey Mpl"), and mouse Mpl-expressing CHO cell line
(hereinafter abbreviated as "CHO-mouse Mpl" ) were established through
25 selection.
1.3 Preparation of soluble human Mpl protein
To prepare soluble human Mpl protein, an expression system using
insect Sf9 cells for production and secretion of the protein was
30 constructed as described below.
A DNA construct encoding the extracellular region of human Mpl
(Gin 26 to Trp 4 91) with a downstream FLAG tag was prepared. The
construct was inserted into a pBACSurf-1 Transfer Plasmid (Novagen)
between the PstI and Smal sites to prepare pBACSurf 1-hMpl-FLAG . Then,
35 Sf9 cells were transformed with 4 |ig of pBACSurf 1-hMpl- FLAG using
the Bac-N-Blue Transfection Kit (Invitrogen) . The culture
55
supernatant was collected after a three-day incubation. Recombinant
virus was isolated by plaque assays. The prepared virus stock was
used to infect Sf9 cells, and the culture supernatant was collected.
Soluble human Mpl protein was purified from the obtained culture
5 supernatant as described below. The culture supernatant was loaded
onto a Q Sepharose Fast Flow (Amersham Biosciences) for adsorption,
and the adsorbed protein was then eluted with 50 mM Na-phosphate buffer
(pH7.2) containing 0.01% (v/v) Tween 20 and 500 mM NaCl. After the
eluates were loaded onto a FLAG M2-Agarose (Sigma-Aldrich) for
10 adsorption, the protein adsorbed was eluted with 100 mM glycine-HCl
buffer (pH3.5) containing 0.01% (v/v) Tween 20. Immediately after
elution, the fraction obtained was neutralized with 1 M Tris-HCl
Buffer (pH8.0) and the buffer was exchanged with PBS(-) and 0.01%
(v/v) Tween 20 using PD-10 columns (Amersham Biosciences) . The
15 purified soluble Mpl protein was referred to as "shMpl-FLAG" .
1.4 Preparation of human Mpl-IgG Fc fusion protein
Human fusion protein Mpl-IgG Fc gene was prepared according to
the method by Bennett et al. (Bennett, B. D. et al., J. Biol. Chem.
20 266, 23060-23067 (1991) ) . A nucleotide sequence encoding the
extracellular region of human Mpl (Gin 26 to Trp 491) was linked to
a nucleotide sequence encoding the Fc region of human IgG-yl (a region
downstream of Asp 216) . A BstEII sequence (amino acids: Val-Thr) was
attached to the junction as a fusion linker between these two regions.
25 A 19-amino acid signal peptide derived form human IgG H chain variable
region was used as the signal sequence. The resulting human fusion
protein Mpl-IgG Fc gene was cloned into pCXND3 to construct
pCXND3-hMpl-Fc.
The vector thus prepared (25 ug) was mixed with a PBS suspension
30 of CHO-DG44 cells (lx 10 7 cells/mL) in Gene Pulser cuvettes. The
mixture was then pulsed at 1.5 kV and 25 uFD using Gene Pulser II
(Bio-Rad) . The CHO cells introduced with the DNA by electroporation
were added to CHO-S-SFMII medium containing 500 ug/mL Geneticin and
lx HT (Invitrogen) . shMPL-Fc-expressing CHO cell line (CHO-hMpl-Fc)
35 was then established through selection.
Human Mpl-IgG Fc fusion protein was purified from the culture
56
supernatant as described below.
The culture supernatant was loaded onto a Q Sepharose Fast Flow
(Amersham Biosciences) for adsorption, and then the adsorbed protein
were eluted with 50 mM Na-phosphate buffer (pH7.6) containing 0.01%
(v/v) Tween 20 and 1 M NaCl. After the eluates were loaded onto a
HiTrap protein G HP column (Amersham Biosciences) for adsorption,
the adsorbed protein was eluted with 0.1 M glycine-HCl buffer (pH2.7)
containing 150 mM NaCl and 0.01% (v/v) Tween 20. Immediately after
elution, the obtained fraction was neutralized with 1 M Tris-HCl
Buffer (pHB.0) and the buffer was exchanged with PBS(-) and 0.01%
(v/v) Tween 20 using PD-10 columns (Amersham Biosciences) . The
purified soluble Mpl protein was referred to as "hMpl-Fc".
1.5 Immunization with shMpl-FLAG or BaF3-human Mpl and hybridoma
selection
MRL/MpJUmmCrj-lpr/lpr mice (hereinafter abbreviated as
"MRL/lpr mice"; purchased from Charles River, Japan) were immunized;
the primary immunization was carried out at eight weeks of age. For
every single mouse, an emulsion containing 100 ug of shMPL-FLAG
combined with Freund' s complete adjuvant (H37 Ra; Beckton Dickinson) ,
was administered subcutaneously as the primary injection. As a
booster injection, an emulsion containing shMPL-FLAG (50 fig per mouse)
combined with Freund' s incomplete adjuvant (Beckton Dickinson) was
administered subcutaneously. Three mice which have been immunized
six times in total were subjected to a final injection of shMPL-FLAG
(50 |ag per mouse) through the caudal vein. Cell fusion was achieved
by mixing the mouse myeloma P3-X63Ag8Ul cells (P3U1; purchased from
ATCC) and mouse splenocytes using polyethylene glycol 1500 (Roche
Diagnostics) . Hybridoma selection in HAT medium began the following
day and culture supernatants were obtained. Screening was carried
out by ELISA, using immunoplates immobilized with shMpl-FLAG or
hMpl-Fc and the assayed cell growth activity of BaF3-human Mpl as
an index. In addition, Balb/C mice were immunized eleven times in
total by administering BaF3-human Mpl (l.Ox 10 7 cells per mouse)
intraperitoneally over a period of one week to five months.
Hybridomas were similarly prepared by cell fusion, and screened using
57
the assayed cell growth activity of BaF3-human Mpl as an index.
Positive clones were isolated as single clones by limiting dilution
and then cultured in a large scale. The culture supernatants were
collected.
5
1.6 Analyses of anti-human Mpl antibodies
Antibody concentrations were determined by carrying out a mouse
IgG sandwich ELISA using goat anti-mouse IgG (gamma) (ZYMED) and
alkaline phosphatase-goat anti-mouse IgG (gamma) (ZYMED), generating
10 a calibration curve by GraphPad Prism (GraphPad Software; USA) , and
calculating the antibody concentrations from the calibration curve.
Commercially available antibodies of the same isotype were used as
standards .
Antibody isotypes were determined by antigen-dependent ELISA
15 using isotype-specif ic secondary antibodies. hMpl-Fc was diluted to
1 Ug/mL with a coating buffer (0.1 mM NaHCOj, pH9.6) containing 0 . 02%
(w/v) NaNa, and then added to ELISA plates . The plates were incubated
overnight at 4°C for coating. The plates were blocked with a diluent
buffer (50 mM Tris-HCl (pH8.1) containing 1 mM MgCl 2 , 150 mM NaCl,
20 0.05% (v/v) Tween 20, 0.02% (w/v) NaN 3 , 1% (w/v) BSA) . After the
addition of hybridoma culture supernatants, the plates were allowed
to stand at room temperature for 1 hr. After washing with a rinse
buffer (0.05% (v/v) Tween 20 in PBS), alkaline phosphatase-labeled
isotype-specif ic secondary antibodies were added to the plates. Then,
25 the plates were allowed to stand at room temperature for 1 hr. Color
development was carried out using SIGMA104 ( Sigma-Aldrich) diluted
to 1 mg/mL with a substrate buffer (50 mM NaHC0 3 , pH9.8) containing
10 mM MgCl2/ and absorbance was measured at 405 nm using Benchmark
Plus (Bio-Rad) .
30 The binding activities of an antibody to shMpl-FLAG and hMPL-Fc
were determined by ELISA. ELISA plates were coated with 1 Ug/mL of
purified shMpl-FLAG or hMPL-Fc, and blocked with a diluent buffer.
Hybridoma culture supernatants were added to the plates, and the
plates were allowed to stand at room temperature for 1 hr. Then,
35 alkaline phosphatase-labeled anti-mouse IgG antibodies (Zymed) were
added to the plates. Color development was similarly carried out
58
using the above method. Following a one-hour coloring reaction at
room temperature, absorbance was measured at 405 nm and EC50 values
were computed using GraphPad Prism.
CHO-human Mpl cells and CHO-monkey Mpl cells were harvested,
and suspended in FACS Buffer (1% FBS/ PBS) to a final concentration
of lx 10 6 cells /mL. The suspensions were aliquoted into Multiscreen
(Millipore) at 100 ul/well, and the culture supernatants were removed
by centrifugation. Culture supernatants diluted to 5 fig/mL were
added to the plates and incubated on ice for 30 min. The cells were
washed once with FACS buffer, and incubated on ice for 30 min following
the addition of an FITC-labeled anti-mouse IgG antibody (Beckman
Coulter) . After incubation, the mixture was centrifuged at 500 rpm
for 1 min. The supernatants were removed, and then the cells were
suspended in 400 UL of FACS buffer. The samples were analyzed by flow
cytometry using EPICS ELITE ESP (Beckman Coulter) . An analysis gate
was set on the forward and side scatters of a histogram to include
viable cell populations.
Agonistic activities of an antibody were evaluated using
BaF3-human Mpl and BaF3-monkey Mpl which proliferate in a
TPO-dependent manner. Cells of each cell line were suspended at 4x
10 5 cells/ml in RPMI 1640/10% FBS (Invitrogen) , and each suspension
was aliquoted into a 96-well plate at 60^1 /well. A 40-\ih aliquot of
rhTPO (R&D) and hybridoma culture supernatants prepared at various
concentrations was added into each well. The plates were then
incubated at 37°C under 5% C0 2 for 24 hr. A 10-uL aliquot of the Cell
Count Reagent SF (Nacalai Tesque) was added into each well. After
incubation for 2 hr, absorbance was measured at 4 50 nm (and at 655
nm as a control) using a Benchmark Plus. EC50 values were calculated
using GraphPad Prism.
The above analysis yielded a total of 163 clones of mouse
monoclonal antibodies that bind to human Mpl.
Among the anti-human Mpl antibodies to be described, TR136 was
established from mice immunized with BaF3-human Mpl and the others
were established from mice immunized with shMpl-Flag.
1.7 Purification of anti-human Mpl antibodies
59
Anti-human Mpl antibodies were purified from hybridoma culture
supernatants as described below.
After the culture supernatants were loaded onto HiTrap protein
G HP columns (Amersham Biosciences) for adsorption, the antibodies
5 were eluted with 0.1 M glycine-HCl <pH2. 7) Buffer. Immediately after
elution, the fractions were neutralized with 1 M Tris-HCl Buffer
(pH9.0), and dialyzed against PBS to replace the buffer for one day.
1.8 Determination of epitopes for the anti-human Mpl antibody VB22B
10 Since the anti-human Mpl antibody VB22B can be used for Western
blotting, a GST-fusion protein containing a partial sequence of human
Mpl was constructed for VB22B epitope analysis. MG1 (Gln26 to Trp491 )
and MG2 (Gln26 to Leu274) regions were each amplified by PCR, and
cloned into pGEX-4T~3 (Amersham Biosciences) to be expressed as GST
15 fusion proteins. The resulting plasmid DNAs were transformed into
DHSa to give transf ormants . A final concentration of 1 mM IPTG was
added to the transf ormants in their logarithmic growth phase to induce
the expression of GST fusion proteins. The bacterial cells were
harvested after two hours of incubation. The cells were lysed by
20 sonication. The lysates were centrifuged in XL-80 Ultracentrif uge
(Beckman, Rotor 70.1Ti) at 35,000 rpm for 30 min. The culture
supernatants were removed, and then the fusion proteins were purified
using GST Purification Modules (Amersham Biosciences) . The samples
were separated by 1 0%-SDS-PAGE, and then transferred onto a PVDF
25 membrane. The membrane was analyzed by the murine antibody VB22B in
Western Btotting. VB22B was found to recognize both MG-1 and MG-2,
indicating that the VB22B epitope is located in the (Gln26 to Leu274)
region .
Then, GST fusion proteins containing the respective regions of
30 human Mpl: MG3 (Gln26 to Alal89) , MG4 (Gln26 to Prol06) , MG5 (Gln26
to Glu259) , and MG6 (Gln2S to Gly245) were prepared and analyzed by
Western blotting using the same procedure described above. VB22B was
found to recognize MG5 and MG6, but not MG3 and MG4 . This suggests
that the VB22B epitope is located within the (Alal89 to Gly245) region.
35 In addition, GST was fused with MG7 (Gln26 to Ala231) and MG8 (Gln26
to Pro217) to prepare GST fusion proteins. VB22B recognized MG7 but
60
not MG8, suggesting that the VB22B epitope is located in the (Gln217
to Ala231) region. Furthermore, GST fusion protein containing MG10
(Gln213 to Ala231) was recognized by VB22B, suggesting that the VB22B
epitope is located within the limited region of 19 amino acids between
5 Gln213 and Ala231.
1.9 Kinetic analyses of the antigen-antibody reaction for anti-human
Mpl antibody VB22B
Since the anti-human Mpl antibody VB22B binds to soluble
10 recombinant Mpl, kinetic analyses of the antigen-antibody reaction
between VB22B IgG and human Mpl-IgG Fc fusion protein were carried
out as described in Example 1.4. The Sensor Chip CM 5 (Biacore) was
placed in Biacore 2000 (Biacore) , and human Mpl-IgG Fc fusion protein
was immobilized onto the chip by amine-coupling methods. Then, 1.25
15 to 20 ug/mL of VB22B IgG solution was prepared using HBS-EP Buffer
(Biacore), and injected over the chip surface for 2 min to reveal
the binding region. Then, HBS-EP Buffer was injected over the chip
surface for 2 min to reveal the dissociation region. VB22B IgG bound
to the human Mpl-IgG Fc fusion protein on the sensor chip was removed
20 by injecting 10 mM NaOH over the sensor chip for 15 sec, and the chip
was recovered. HBS-EP Buffer was used as the running buffer, and the
flow rate was 20 nL/min. Using the BIAevaluation Version 3.1
(Biacore) software, the reaction rate constant at each concentration
was calculated from the sensorgrams. The dissociation constant (KD)
25 for VB22B IgG was determined to be 1.67 ± 0.713 x 10" 9 M.
[Example 2] Preparation of single-chain anti-human Mpl antibodies
Among the prepared anti-human Mpl antibodies, 23 types of
antibodies, which exhibit higher binding activities and agonistic
30 activities, were selected to construct expression systems for
single-chain antibodies using genetic engineering technigues. An
exemplary method for constructing a single-chain antibody derived
from the anti-human Mpl antibody VB22B is described below.
35 2.1 Cloning of the anti-human Mpl antibody variable region
The variable region was amplified by RT-PCR using total RNA
61
extracted from hybridomas producing anti-human Mpl antibodies.
Total RNA was extracted from lx 10 7 hybridoma cells using the RNeasy
Plant Mini Kit (QIAGEN) .
A 5' -terminal fragment of the gene was amplified from 1 ug of
5 total RNA by the SMART RACE cDNA Amplification Kit (Clontech) , using
a synthetic oligonucleotide MHC-IgG2b (SEQ ID NO: 166) complementary
to mouse IgG2b constant region or a synthetic oligonucleotide kappa
(SEQ ID NO: 167) complementary to mouse k chain constant region.
Reverse transcription was carried out at 42°C for 1.5 hr .
10 The composition of the PCR reaction solution (50 (1L in total)
is shown below.
lOx Advantage 2 PCR Buffer (Clontech)
5
ML
lOx Universal Primer A Mix (Clontech)
5
HL
dNTPs (dATP, dGTP, dCTP, and dTTP) (Clontech)
0
2
mM
Advantage 2 Polymerase Mix (Clontech)
1
ML
Reverse transcription product
2
5
ML
Synthetic oligonucleotide, MHC-IgG2b or kappa
10
pmol
The PCR reaction conditions were:
94°C (initial temperature) for 30 sec;
15 five cycles of 94°C for 5 sec and 72°C for 3 min;
five cycles of 94 °C for 5 sec, 70°C for 10 sec, and 72°C for 3 min;
25 cycles of 94°C for 5 sec, 68°C for 10 sec, and 72°C for 3 min;
and final extension was at 72°C for 7 min.
The PCR products were purified from agarose gel using the
20 QIAquick Gel Extraction Kit (QIAGEN), and cloned into a pGEM-T Easy
Vector (Promega) . The nucleotide sequence was then determined using
the ABI 3700 DNA Analyzer (Perkin Elmer).
The nucleotide sequence of cloned VB22B H chain variable region
(hereinafter abbreviated as "VB22B-VH" ) is shown in SEQ ID NO: 117,
25 and its amino acid sequence is shown in SEQ ID NO: 118. The nucleotide
sequence of the L chain variable region (hereinafter abbreviated as
"VB22B-VL") is shown in SEQ ID NO: 119, and its amino acid sequence
is shown in SEQ ID NO: 120.
62
2.2 Preparation of expression vectors for anti-human Mpl diabodies
The gene encoding VB22B single-chain Fv (hereinafter
abbreviated as "VB22B diabody") containing a five-amino acid linker
sequence was constructed, by linking a nucleotide sequence encoding
5 a (Gly«Ser)i linker to the VB22B-VH-encoding gene at its 3' end and
to the VB22B-VL-encoding gene at its 5' end; both of which have been
amplified by PCR.
The VB22B-VH forward primer, 70-115HF, ( SEQ ID NO: 168) was
designed to contain an EcoRI site. The VB22B-VH reverse primer,
10 33-115HR, (SEQ. ID NO: 169) was designed to hybridize to a DNA encoding
the C terminus of VB22B-VH, and to have a nucleotide sequence encoding
the (Gly^Ser) i linker and a nucleotide sequence hybridizing to the
DNA encoding the N terminus of VB22B-VL. The VB22B-VL forward primer,
33-115LF, (SEQ ID NO: 170) was designed to have a nucleotide sequence
15 encoding the N terminus of VB22B-VL, a nucleotide sequence encoding
the (Gly 4 Ser)i linker, and a nucleotide sequence encoding the C
terminus of VB22B-VH. The VB22B-VL reverse primer, 33-115LR, (SEQ
ID NO: 171) was designed to hybridize to a DNA encoding the C terminus
of VB22B-VL and to have a nucleotide sequence encoding a FLAG tag
20 (Asp Tyr Lys Asp Asp Asp Asp Lys/SEQ ID NO: 172) and a NotI site.
In the first round of PCR, two PCR products: one containing
VB22B-VH and a linker sequence, and the other containing VB22B-VL
and the identical linker sequence, were synthesized by the procedure
described below.
25 The composition of the PCR reaction solution (50 uL in total)
is shown below.
lOx PCR Buffer (TaKaRa)
5 UL
dNTPs (dATP, dGTP, dCTP, and dTTP) (TaKaRa)
0 . 4 mM
DNA polymerase TaKaRa Ex Taq (TaKaRa)
2 . 5 units
pGEM-T Easy vector comprising VB22B-VH or
VB22B-VL gene
10 ng
Synthetic oligonucleotides, 70-115HF and
33-115HR, or 33-115LF and 33-115LR
10 pmol
The PCR reaction conditions were:
63
94°C (initial temperature) for 30 sec-
five cycles of: 94°C for 15 sec and 72°C for 2 min;
five cycles of 94 °C for 15 sec and 70°C for 2 min;
28 cycles of 94 °C for 15 sec and 68°C for 2 min;
5 and final extension was at 72°C for 5 min.
After the PCR products of about 400 bp were purified from agarose
gel using the QIAquick Gel Extraction Kit (QIAGEN), the second-round
PCR was carried out using aliquots of the respective PCR products
according to the protocol described below.
10 The composition of the PCR reaction solution (50 uL in total)
is shown below.
lOx PCR Buffer (TaKaRa)
5 UL
dNTPs (dATP, dGTP, dCTP,
and dTTP) (TaKaRa)
0
4 mM
DNA polymerase TaKaRa Ex
Taq (TaKaRa)
2 . 5
unit
First-round PCR products
(two types)
1 UL
Synthetic oligonucleotides, 70-115HFand 33-115LR
10
pmol
The reaction conditions were:
94°C (initial temperature) for 30 sec;
15 five cycles of 94°C for 15 sec and 72°C for 2 min;
five cycles of 94 °C for 15 sec and 70°C for 2 min;
28 cycles of 94 °C for 15 sec and 6B°C for 2 min;
and final extension was at 72 °C for 5 min.
The PCR products of about 800 bp were purified from agarose gel
20 using the QIAquick Gel Extraction Kit (QIAGEN), and then digested
with EcoRI and NotI (both from TaKaRa) . The resulting DNA fragments
were purified using the QIAquick PCR Purification Kit (QIAGEN) , and
then cloned into pCXND3 to prepare pCXND3-VB22B db.
25 2.3 Preparation of expression vectors for anti-human Mpl antibody
sc (Fv) 2
To prepare expression plasmids for the modified antibody
[sc(Fv)2] comprising two units of H chain variable region and two
units of L chain variable region derived from VB22B, the
30 above-described pCXND3-VB22B db was modified by PCR using the
64
procedure shown below . The process for constructing the sc ( Fv) 2 gene
is illustrated in Fig. 1.
First, PCR method was carried out to amplify (a) the
VB22B-VH-encoding gene in which a nucleotide sequence encoding a
5 15-amino acid linker (Gly 4 Ser) 3 was added to its 3' end; and (b) the
VB22B-VL-encoding gene containing the identical linker nucleotide
sequence added to its 5' end. The desired construct was prepared by
linking these amplified genes. Three new primers were designed in
this construction process'. The VB22B-VH forward primer, VB22B-fpvu,
10 (primer A; SEQ ID NO: 173) was designed to have an EcoRI site at its
5' end and to convert Gln22 and Leu23 of VB22B db into a PvuII site.
The VB22B-VH reverse primer, sc-rL15, (primer B; SEQ ID NO: 174) was
designed to hybridize to a DNA encoding the C terminus of VB22B-VH,
and to have a nucleotide sequence encoding the (Gly4Ser) 3 linker, as
15 well as a nucleotide sequence hybridizing to a DNA encoding the N
terminus of VB22B-VL. The VB22B-VL forward primer, sc-fL15, (primer
C; SEQ ID NO: 175) was designed to have a nucleotide sequence encoding
the N terminus of VB22B-VL, a nucleotide sequence encoding the
(Gly 4 Ser) 3 linker, and a nucleotide sequence encoding the C terminus
20 of VB22B-VH.
In the first-round PCR, two PCR products: one comprising
VB22B-VH and a linker sequence, and the other comprising VB22B-VL
and the identical linker sequence, were synthesized by the procedure
described below.
25 The composition of the PCR reaction solution (50 UL in total)
is shown below.
lOx PCR Buffer (TaKaRa)
5 \IL
dNTPs (dATP, dGTP, dCTP, and dTTP) (TaKaRa)
0.4 mM
DNA polymerase TaKaRa Ex Taq (TaKaRa)
2 . 5 units
pCXND3-VB22B db
10 ng
Synthetic oligonucleotides, VB22B-fpvu, sc-rL15
10 pmol
or sc-fL15, and 33-115LR (primer D)
The reaction conditions were:
94 °C (initial temperature) for 30 sec;
65
five cycles of 94°C for 15 sec and 72°C for 2 min;
five cycles of 94 °C for 15 sec and 70°C for 2 min;
28 cycles of 94°C for 15 sec and 68°C for 2 min;
and final extension was at 72°C for 5 rain.
5 After the PCR products of about 400 bp were purified from agarose
gel using the QIAquick Gel Extraction Kit (QIAGEN), the second-round
PCR was carried out using aliquots of the respective PCR products
according to the protocol described below.
The composition of the PCR reaction solution (50 UL in total)
10 is shown below.
lOx PCR Buffer (TaKaRa)
5 UL
dNTPs (dATP, dGTP, dCTP, and dTTP) (TaKaRa)
0.4 mM
DNA polymerase TaKaRa Ex Taq (TaKaRa)
2 . 5 units
First-round PCR product (two types)
1 UL
Synthetic oligonucleotide, 70-115HF and 33-115LR
10 pmol
The reaction conditions were:
94°C (initial temperature) for 30 sec-
five cycles of 94 °C for 15 sec and 72°C for 2 min;
15 five cycles of 94°C for 15 sec and 70°C for 2 min;
28 cycles of 94 °C for 15 sec and 68°C for 2 min;
and final extension was at 72°C for 5 min.
The PCR products of about 800 bp were purified from agarose gel
using the QIAquick Gel Extraction Kit (QIAGEN) , and then digested
20 with EcoRI and NotI (both from TaKaRa) . The resulting DNA fragments
were purified using the QIAquick PCR Purification Kit (QIAGEN) , and
then cloned into pBacPAK9 (Clontech) to construct pBacPAK9-scVB22B .
A fragment to be inserted into the PvuII site of
pBacPAK9-scVB22B was prepared. Specifically, the fragment has a
25 PvuII recognition site at both ends and a nucleotide sequence, in
which a gene encoding the VB22B-VH N-terminus is linked, via a
(Gly.iSer)3 linker-encoding nucleotide sequence, to a gene encoding
the amino acid sequence of an N terminus-deleted VB22B-VH linked to
VB22B-VL via the (Gly^Ser^ linker. Two primers were newly designed
30 to prepare the fragment by PCR. The forward primer for the fragment
66
of interest, Fv2-f (primer E; SEQ ID NO: 176), was designed to have
a PvuII site at its 5' end and a VB22B-VH 5' -end sequence. The reverse
primer for the fragment of interest, Fv2-r (primer F; SEQ ID NO: 177) ,
was designed to hybridize to a DNA encoding the C terminus of VB22B-VL,
5 and to have a PvuII site, a nucleotide sequence encoding the (Gly«Ser) 3
linker, and a nucleotide sequence hybridizing to a DNA encoding the
N terminus of VB22B-VH. PCR was carried out using pBacPAK9-scVB22B
as a template as described below.
The composition of the PCR reaction solution (50 UL in total)
10 is shown below.
lOx PCR Buffer (TaKaRa)
5 UL
dNTPs (dATP, dGTP, dCTP, and dTTP) (TaKaRa)
0.4 mM
DNA polymerase TaKaRa Ex Taq (TaKaRa)
2 . 5 units
pBacPAK9-scVB22B
10 ug
Synthetic oligonucleotide, Fv2-f and Fv2-r
10 pmol
The reaction conditions were:
94 °C (initial temperature) for 30 sec-
five cycles of 94°C for 15 sec and 72°C for 2 min;
15 five cycles of 94°C for 15 sec and 70 D C for 2 min;
28 cycles of 94 °C for 15 sec and 68°C for 2 min;
and final extension was at 72°C for 5 min.
The PCR products of about 800 bp were purified from agarose gel
using the QIAquick Gel Extraction Kit (QIAGEN) , and then cloned into
20 the pGEM-T Easy Vector (Promega) . After sequencing, the plasmid was
digested with PvuII (TaKaRa) , and the fragment of interest was
recovered. The recovered fragment was ligated to pBacPAK9-scVB22B
pre-digested with PvuII (TaKaRa) to construct pBacPAK9-VB22B sc (Fv) 2 .
After the resulting vector was digested with EcoRI and NotI (both
25 from TaKaRa) , the fragment of about 1, 600 bp was purified from agarose
gel using the QIAquick Gel Extraction Kit (QIAGEN) . The fragment was
then cloned into a pCXND3 expression vector to construct pCXND3-VB22B
sc (Fv) 2.
30 2.4 Expression of single-chain anti-human Mpl antibody in animal cells
67
A cell line stably expressing the single-chain antibody was
prepared from CHO-DG44 cells as described below. Gene transfer was
achieved by electroporation using a Gene Pulser II (Bio-Rad) . An
expression vector (25 ug) and 0.75 mL of CHO-DG44 cells suspended
in PBS (Ix 10 7 cells/mL) were mixed. The resulting mixture was cooled
on ice for 10 min, transferred into a cuvette, and pulsed at 1.5-kV
and 25 fiFD. After a ten-minute restoration period at room temperature,
the electroporated cells were plated in CHO-S-SFMIX medium
(Invitrogen) containing 500 Hg/mL Geneticin (Invitrogen) . CHO cell
lines expressing the single-chain antibody were established through
selection. A cell line stably expressing VB22B sc(Fv)2 and its
culture supernatants were obtained by this method.
The transient expression of the single-chain antibody was
achieved using COS7 cells as described below. An expression vector
(10 ug) and 0.75 mL of COS7 cells suspended in PBS <lx 10 7 cells/mL)
were mixed. The resulting mixture was cooled on ice for 10 min,
transferred into a cuvette, and then pulsed at 1.5-kV and 25 M-FD .
After a ten-minute restoration period at room temperature, the
electroporated cells were plated in DMEM/10% FBS medium (Invitrogen) .
The cells were incubated overnight and then washed with PBS.
CHO-S-SFMII medium was added and the cells were cultured for about
three days. The culture supernatants for preparing the VB22B diabody
were thus prepared.
2. 5 Quantitation of single-chain anti-human Mpl antibodies in culture
supernatant s
The culture supernatant concentration of the single-chain
anti-human Mpl antibody transiently expressed in COS cells was
determined using surface plasmon resonance. A sensor chip CM 5
(Biacore) was placed in Biacore 2000 (Biacore) . ANTI-FLAG® M2
Monoclonal Antibody ( Sigma-Aldrich) was immobilized onto the chip.
An appropriate concentration of sample was injected over the chip
surface at a flow rate of 5 mL/sec, and 50 mM diethylamine was used
to dissociate the bound antibody. Changes in the mass during sample
injection were recorded, and the sample concentration was calculated
from the calibration curve prepared using the mass changes of a
68
standard sample. dbl2E10 (see WO 02/33073 and WO 02/33072) was used
as the diabody standard, and 12E10 sc(Fv)2 which has the same gene
structure as that of sc(Fv)2 was used as the sc(Fv)2 standard.
5 2.6 Purification of anti-human Mpl diabodies and single-chain
antibodies
The culture supernatants of VB22B diabody-expressing COS7 cells
or CHO cells was loaded onto an Anti-Flag M2 Affinity Gel
( Sigma-Aldrich) column equilibrated with a 50 mM Tris-HCl buffer
10 (pH7.4) containing 150 mM. NaCl and 0.05% Tween 20. The absorbed
antibodies were eluted with 100 mM glycine-HCl (pH3.5). The
fractions eluted were immediately neutralized with 1 M Tris-HCl
(pH8.0), and loaded onto a HiLoad 26/60 Superdex 200 pg (Amersham
Biosciences) column for gel filtration chromatography. PBS/0.01%
15 Tween 20 was used in the gel filtration chromatography.
VB22B sc(Fv)2 was purified from the culture supernatants of
VB22B sc (Fv) 2-expressing COS7 cells or CHO cells under the same
conditions used for purifying the diabodies. A large-scale
preparation of VB22B sc(Fv)2 was prepared by loading the CHO cell
20 culture supernatants onto a Macro-Prep Ceramic Hydroxyapatite Type
I (Bio-Rad) column equilibrated with a 20 mM phosphate buffer (pH6.8),
and eluting the VB22B sc(Fv)2 in a stepwise manner with 250 mM
phosphate buffer (pH6.8). The eluted fraction was concentrated on
an ultrafilter, and then fractionated by gel filtration
25 chromatography using a HiLoad 26/60 Superdex 200 pg (Amersham
Biosciences) column, and a fraction corresponding to the molecular
weight range of about 40 kD to 70 kD was obtained. The fraction was
loaded onto an Anti-Flag M2 Affinity Gel column equilibrated with
a 50 mM Tris-HCl buffer (pH7.4) containing 150 mM NaCl and 0.05% Tween
30 20. The absorbed antibody was eluted with 100 mM glycine-HCl (pH3.5).
The eluted fraction was immediately neutralized with 1 M Tris-HCl
(pH8.0), and loaded onto a HiLoad 26/60 Superdex 200 pg (Amersham
Biosciences) column for gel filtration chromatography . 20 mM acetate
buffer (pH6.0) containing 150 mM NaCl and 0.01% Tween 80 was used
35 in the gel filtration chromatography. In each purification step, the
presence of the diabody and sc(Fv)2 in the samples was confirmed by
69
SDS-PAGE and Western blotting using an anti-Flag antibody
(Sigma-Aldrich) . Specifically, obtained fractions corresponding to
each peak were subjected to the electrophoresis according to the
method described by Laemli, and then stained using Coomassie Brilliant
5 Blue. As a result, single band was detected apparently at about 29
kDa for the diabody; while single band was detected apparently at
about 55 kDa for sc(Fv)2.
2.7 Binding activity analyses of single-chain anti-human Mpl
10 antibodies by flow cytometry
CHO-human Mpl, CHO-monkey Mpl, and CHO-mouse Mpl cells were
recovered and suspended in FACS buffer (1% FBS/PBS) to a final
concentration of Ix 10 6 cells/mL. Cell suspensions were aliquoted
at 100-^L/well into the Multiscreen-HV Filter Plates (Millipore) .
15 After centrif ugation, the supernatant was removed. An appropriate
concentration of diabody or sc<Fv)2 was added into each well and
incubated on ice for 30 min. The cells were washed once with 200 uL
of FACS buffer, and incubated on ice for 30 min following the addition
of 10 U-g/mL ANTI-FLAG® M2 Monoclonal Antibody (Sigma-Aldrich) . The
20 cells were then washed once with 200 UL of FACS buffer, and a
lOOx-diluted FITC-labeled anti-mouse IgG antibody (Beckman Coulter)
was added to the plate. The plate was incubated on ice for 30 min.
After centrifugation, the supernatant was removed. The cells were
suspended in 400 uL of FACS Buffer, and then analyzed by flow cytometry
25 using EPICS ELITE ESP (Beckman Coulter) . An analysis gate was set
on the forward and side scatters of a histogram to include viable
cell populations.
The binding activity of the purified VB22B sc(Fv)2 to various
Mpl molecules expressed in CHO cells was determined (Fig. 2) . VB22B
30 sc (Fv) 2 was found to specifically bind to CHO-human Mpl and CHO-monkey
Mpl but not to the host cell CHO or CHO-mouse Mpl. This binding
characteristic of VB22B sc(Fv)2 is comparable to those of VB22B IgG,
indicating that the antibody binding site remains unaltered by
converting whole IgG to minibody.
35
2.8 Analyses of TPO-like agonistic activity for single-chain
70
anti-human Mpl antibodies
TPO-like agonistic activity was assessed using BaF3-human Mpl
or BaF3-monkey Mpl that proliferate in a TPO-dependent manner.
Cells from each cell line were washed twice with RPMI 1640/1%
FBS (fetal bovine serum) ( Invitrogen) , and then suspended in RPMI
1640/10% FBS to a concentration of 4x 10 5 cells/mL. Cell suspensions
were aliquoted at 60-uL/well into a 96-well plate. Various
concentrations of rhTPO (R&D) and COS7 culture supernatants or
purified samples were prepared, and a 4 0-uL aliquot was added into
each well. The plates were then incubated at 37 °C under 5% C0 2 for
24 hr. Immediately after a 10-uL aliquot of WST-8 reagent (Cell Count
Reagent SF; Nacalai Tesque) was added into each well, absorbance was
measured at 450 nm (and at 655 ran as a control) using Benchmark Plus.
After two hours of incubation, absorbance was again measured at 450
nm (and at 655 nm as a control) . The WST-8 reagent changes colors
at 450 nm in a color reaction that reflects the viable cell count.
The TPO-like agonistic activity was assessed using the change in
absorbance during the two-hour incubation as an index. EC 5 o values
were computed using GraphPad Prism.
TPO-like agonistic activity was assayed using the human
leukemia cell line M-07e (purchased from DSMZ) which proliferates
TPO-dependently. M-07e cells were washed twice with RPMI 1640/1% FBS,
and then suspended in RPMI 1640/10% FBS to a concentration of 5x 10 s
cells/mL. The resulting cell suspension was aliquoted at 50-uL/well
into a 96-well plate. Various concentrations of rhTPO and COS7
culture supernatants or purified samples were prepared, and a 50-ui
aliquot was added into each well. The plates were then incubated at
37°C under 5% C0 2 for 48 hr. Immediately after a 10-uL aliquot of
WST-8 reagent (Cell Count Reagent SF; Nacalai Tesque) was added to
each well, absorbance of was measured at 450 nm (and at 655 nm as
a control) using a Benchmark Plus. After four hours of incubation,
absorbance was again measured at 450 nm (and at 655 nm as a control) .
The TPO-like agonistic activity was assayed using the change in
absorbance during the four-hour incubation as an index.
Purified VB22B IgG, VB22B diabody, and VB22B sc(Fv)2 were
assayed for their TPO-like agonistic activities using BaF3-human Mpl,
71
BaF3-monkey Mpl, and M-07e. The results are shown in Figs. 3, 4, and
5, respectively. The presence of bivalent antigen-binding domains
in a single antibody molecule is essential for its agonistic activity.
The distance and angle between two antigen-binding domains can also
be important factors (see WO 02/33073 and WO 02/33072). Similar
results were obtained for the newly isolated anti-human Mpl antibodies .
Specifically, the agonistic activities of VB22B diabody and VB22B
sc(Fv)2 (EC 50 = 61 pM and 27 pM in BaF3-human Mpl, respectively) were
higher than that of VB22B IgG (EC 5 o > 30 nM in BaF3-human Mpl), and
were equivalent to or higher than that of the naturally-occurring
human TPO ligand (EC 50 = 76 pM in BaF3-human Mpl) . The VB22B diabody
activity was lower than that of VB22B sc(Fv)2. This suggests that
the structure of a single-chain antibody is greatly altered by its
molecular shape and the length of the linker sequence, which in turn
changes the agonistic activity. Sixteen types of the single-chain
anti-human Mpl antibodies were obtained, each exhibiting a high
agonistic activity. The amino acid sequences of the H chain and L
chain variable regions of the representative antibodies are shown
in Figs. 6 and 7, respectively.
2.9 Humanization of single-chain anti-human Mpl antibody
Antibody sequence data for the humanization of VB22B sc(Fv)2
were obtained from the Kabat Database
(ftp://ftp.ebi.ac.uk/pub/databases/kabat/), and homology searches
were carried out independently for the H chain variable region and
the L chain variable region. As a result, the H chain variable region
was found to be highly homologous to DN13 (Smithson S. L. et al.,
Mol Immunol. 36, 113-124 (1999)). The L chain variable region was
found to be highly homologous to ToP027 (Hougs L. et al., J. Immunol.
162, 224-237 (1999)). Humanized antibodies were prepared by
inserting a complementarity-determining region (hereinafter
abbreviated as "CDR" ) into the framework regions (hereinafter
abbreviated as "FR") of the above antibodies. The humanized antibody
sc(Fv)2 was expressed in CHO-DG44 cells, and its agonistic activity
was assessed using BaF3-human Mpl. The agonistic activity was used
as an index to generate a humanized VB22B sc(Fv)2 which has agonistic
72
activity equivalent to that of murine VB22B sc(Fv)2 by replacing one
or more amino acids in its framework region.
Specifically, synthetic oligo-DNAs of approximately 50
nucleotides in length were designed as to make 20 of these nucleotides
available for hybridization, and the synthetic oligo-DNAs were
assembled by PCR to prepare genes that encode the respective variable
regions. Using the resulting genes, sc(Fv)2 was similarly prepared
by the method described in Example 2.3. The respective DNAs were
cloned into a pCXND3expression vector to construct expression vectors
pCXND3-hVB22B p-z sc(Fv)2, pCXND3-hVB22B g-e sc(Fv)2, pCXND3-hVB22B
e sc{Fv)2, pCXND3-hVB22B u2-wz4 sc(Fv)2, and pCXND3-hVB22B q-wz5
sc(Fv)2, to which the humanized VB22B sc(Fv)2 is inserted. The
nucleotide sequences and the amino acid sequences of the fragments
in each plasmid are shown below.
Plasmid name Nucleotide Amino acid
sequence sequence
hVB22B p-z sc(Fv)2
SEQ
ID NO
1
SEQ
IC
NO
2
hVB22B g-e sc(Fv)2
SEQ
ID
NO:
253
SEQ
ID
NO:
254
hVB22B e sc (Fv) 2
SEQ
ID
NO:
259
SEQ
ID
NO:
260
hVB22B u2-wz4 sc(Fv)2
SEQ
ID
NO:
286
SEQ
ID
NO:
287
hVB22B q-wz5 sc(Fv)2
SEQ
ID
NO:
292
SEQ
ID
NO:
293
Murine VB22B sc(Fv)2
SEQ
ID
NO:
263
SEQ
ID
NO:
264
The plasmids were expressed in CHO-DG44 cells and the culture
supernatants were recovered by the method described in Example 2.4.
Since the humanized VB22B sc(Fv)2 does not contain a Flag tag, its
purification from the culture supernatant was performed using a
MG10-GST fusion protein. MG10 (Gln213 to Ala231) is one of the
epitopes recognized by VB22B, as described in Example 1.8. The
MG10-GST fusion protein was purified using Glutathione Sepharose 4B
(Amersham Biosciences) according to the supplier's protocol. Then,
the purified MG10-GST fusion protein was immobilized onto a HiTrap
NHS-activated HP Column (Amersham Biosciences ) to prepare an affinity
column, according to the supplier's protocol. The culture
supernatant of CHO cells expressing the humanized VB22B sc(Fv)2 was
73
loaded onto the MG10-GST fusion protein-immobilized column, which
has been equilibrated with 50 mM Tris-HCl (pH7.4)/150 mM NaCl/0.01%
Tween 80. The adsorbed humanized VB22B sc(Fv)2 was eluted with 100
mM glycine-HCl (pH3 . 5 ) /0 . 01% Tween 80. Immediately after elution,
the eluted fraction was neutralized with 1 M Tris-HCl (pH7 . 4 ) , and
was further subjected to gel filtration chromatography using a HiLoad
16/60 Superdex 200 pg (Amersham Biosciences) . 20 mM citrate buffer
(pH7.5) containing 300 mM NaCl and 0.01% Tween 80 was used in the
gel filtration chromatography. The TPO-like agonistic activities of
the purified samples were similarly determined using the method
described in Example 2.8. The TPO-like agonistic activities of the
purified murine VB22B sc(Fv)2, hVB22B p-z sc(Fv)2, hVB22B u2-wz4
sc(Fv)2, hVB22B q-wz5 sc(Fv>2, and humanized hVB22B e sc(Fv}2 and
hVB22B g-e sc'(Fv) 2 were assessed in BaF3-human Mpl . The results are
shown in Figs. 19, 20, and 21. The humanized VB22B sc(Fv)2 showed
comparable agonistic activities, suggesting that the humanization
has no influence on the activity.
2.10 Kinetic analyses of the antigen-antibody reaction for anti-human
Mpl antibodies: VB22B IgG, VB22B sc(Fv)2, and humanized VB22B sc(Fv)2
Using the soluble recombinant Mpl-binding characteristic of
anti-human Mpl antibody VB22B, kinetic analyses of the
antigen-antibody reactions between the MG10 (Gin 213 to Ala 231) -GST
fusion protein and each of VB22B IgG, VB22B sc(Fv)2, and humanized
VB22B sc ( Fv) 2 were carried out as described in Example 1.8. The Sensor
Chip CM5 (Biacore) was placed in Biacore 3000 (Biacore) , and MG10-GST
fusion protein was immobilized onto the chip by amine-coupling methods
HBS-EP Buffer (Biacore) was used as the running buffer, and the flow
rate was 20 uL/min. 5 . 5 to 175 . 0 nM of VB22B IgG solution was prepared
using HBS-EP Buffer, and injected over each of the chip surfaces for
2 min to obtain the binding region at the respective concentrations.
Then, dissociation region for the 2 minutes was measured. VB22B IgG
bound to the MG10-GST fusion protein on the sensor chip was removed
by injecting' 20 mM HC1 over the sensor chip for 1 min, and the chip
was recovered. Similarly, 4.7 to 150.1 nM of VB22B sc(Fv)2, 5.3 to
168. 9 nM of hVB22B q-wz5 sc(Fv)2, and 4. 9 to 156.8 nM of hVB22B u2-wz4
74
sc(Fv)2 were prepared and injected over the chip surfaces onto which
MG10-GST fusion protein was immobilized, and the measurement was
carried out.
All the antibodies used were bivalent antibodies, and thus the
5 sensorgrams at each concentration were obtained in the presence of
both monovalent and bivalent bindings. In this context, the reaction
rate constant was determined as that for the monovalent antibody by
analysis using the Bivalent analyte model of BIAevaluation ver.3.1
software (Biacore) . The above analysis was carried out in
10 triplicates for each antibody. The binding rate constant (ka) ,
dissociation rate constant (kd) , and dissociation constant (KD) were
determined as those for the monovalent antibody by the procedure
described above. The constants are indicated below in Table 1. The
dissociation constants (KD) for VB22B IgG, VB22B sc (Fv) 2, hVB22Bq-wz5
15 sc(Fv)2, and hVB22B u2-wz4 sc(Fv)2 were determined to be 1.15 x 10" 8
M, 1.17 x 10" 8 M, 1.36 x 1CT B M, and 1.02 x 1CT B M, respectively, showing
nearly equivalent binding activities towards the MG10-GST fusion
protein .
20 Table 1 Kinetic analyses of the antigen-antibody reaction for
anti-human Mpl antibodies
Antibody Name
ka (1/Ms)
[xlO 5 ]
kd (1/s)
[xlO" 3 ]
KD (M)
[xlO" 8 ]
VB22B IgG
0.96 ± 0.78
1.10 ± 0.01
1.15 ± 0.09
VB22B sc(Fv)2
4.23 ± 0.22
4.91 ± 0.72
1.17 ± 0.23
hVB22B q-wz5 sc(Fv)2
3.76 ± 0.38
5.10 ± 0.56
1.36 ± 0. 06
hVB22B u2-wz4 sc(Fv)2
6.08 ± 0.30
6.17 ± 0.23
1. 02 ± 0.08
[Example 3] Preparation of anti-Mpl diabodies by the AGS method
Anti-Mpl diabodies having agonistic activity were prepared by
25 an Autocrine Growth Selection (AGS) method (see, WO 03/91424).
3.1 Construction of a retrovirus library
Spleens were isolated from MRL/lpr mice immunized with
shMPL-Flag by the method described in Example 1.5, and homogenized
75
in TRIZOL Reagent (Invitrogen) using a Dounce homogenizer. After
chloroform addition, the homogenized sample was shaken vigorously,
the aqueous phase was removed and total RNA was extracted by
isopropanol precipitation. mRNA was purified using a PolyATract
5 System 1000 (Promega). Reverse transcription of 2.5 Ug mRNA was
carried out at 42°C for 50 min using the Superscript First strand
synthesis system for RT-PCR (Invitrogen) and the included oligo-dT
primers to prepare cDNA.
The composition of the PCR reaction solution (250 uL) is shown
10 below.
lOx KOD Plus Buffer (Toyobo)
25 uL
2 mM dNTPs (dATP, dGTP, dCTP, and dTTP) (Toyobo)
2 5 UL
2.5 mM MgS0 4 (Toyobo)
10 UL
KOD Plus (Toyobo)
7 . 5 UL
Reverse transcription products
2 5 uL
Mixed primers complementary to H chain or L chain
500 pmol
variable region
The reaction conditions were:
98°C (initial temperature) for 3 min;
32 cycles of 98°C for 20 sec, 58°C for 20 sec, and 72°C for 30 sec;
15 and final extension was at 72°C for 6 min.
The H chain primer mix contained HS1 to HS19 (SEQ ID NOs : 178
to 196) and HA1 to HA4 (SEQ ID NOs: 197 to 200), which were mixed
at the indicated ratios next to the sequence names in Table 2. The
L chain primer mix contained LSI to LS17 (SEQ ID NOs: 201 to 217),
20 Lslambda (SEQ ID NO: 218), LAI to LA 5 (SEQ ID NOs: 219 to 222), and
Lalambda ( SEQ ID NO: 223 ) . The respective PCR products were purified
from agarose gel using the QIAquick Gel Extraction Kit (QIAGEN) . The
H chain and L chain variable regions were linked via the (Gly 4 Ser)i
linker sequence by PCR using sc-S (SEQ ID NO; 224) and sc-AS (SEQ
25 ID NO: 225) as described below.
The composition of the PCR reaction solution (100 |iL in total)
is shown below.
lOx KOD Plus Buffer (Toyobo)
10 UL
76
2 mM dNTPs (dATP, dGTP, dCTP,
and dTTP) (Toyobo)
10
2.5 mM MgS0 4 (Toyobo)
4
UL
KOD Plus (Toyobo)
2
UL
Fragment of H chain variable
region
4
Fragment of L chain variable
region
4
UL
The first-round PCR conditions were:
94 "C (initial temperature) for 3 min; and
seven cycles of 94°C for 1 min and 63°C for 4 min.
5 Then, sc-S and sc-AS (25 pmol each) were added to the first-round
products .
The second-round PCR conditions were:
30 cycles of 94°C for 30 sec, 55"C for 2 min, and 72°C for 2 min;
and final extension was at 72°C for 6 min.
10 The resulting product with an Sf il restriction site at both ends
was purified using the QIAquick PCR Purification Kit (QIAGEN), and
incubated with the Sfil restriction enzyme (TaKaRa) overnight at 50 °C .
The PCR product purified from agarose gel using the QIAquick Gel
Extraction Kit (QIAGEN) was inserted into the Sfil site of the viral
15 vector pMX/IL3ssGFPHis .
The resulting plasmid was constructed by inserting a GFP gene,
which has an EcoRI site, mouse IL-3 signal sequence and Sfil site
at its 5' end; and an Sfil site, His tag sequence, termination codon,
and NotI site at its 3' end, between the EcoRI and NotI sites on the
20 pMX viral vector (Onishi, M. etal., Mol. Cell. Biol. 18, 3871-3879).
The plasmid was introduced into the ElectroMAX DH10B Tl phage
resistant cells (Invitrogen) by electroporation (settings: 2.5 kV,
25 UF, and 100Q) using a Gene Pulser II (Bio-Rad) . The cells were
plated onto an LB-Agar plate containing 100 Ug/mL ampicillin. After
25 overnight incubation, lx ID 1 colonies were obtained. Colonies were
recovered from the plate and plasmids were then extracted using the
QIAGEN Plasmid Maxi Kit (QIAGEN) .
77
Table 2
SEQ
ID
NO-
178
[HS1 (4 ) )
BCPrARCCRGCCATGGCGGAKGTRMAGCTTCAGGAGTC
sou
ID
NO;179
(•tie 5 IA\)
GrPfAfVTRRfTATGGCGGAGGTBCAGCTBCAGCAGTC
Tr>
NO
1 80
( / 1 \ }
moj \ j / /
GPPPAraPPGGPPATGGnRPAfi£3TGCAGCTGAAOSAS , rC?
CPA
Tn
NO
181
[ H<3d / A \ ^
tnuv \ * 1 I
fSPPPAriPPfsnPPATGGnnGAGGTPCAJICTGCAACAllTP
epft
XL)
NO
182
(HS5 (7) )
GCCCAGCCGGCCATGGCGCAGGTYCAGCTBCAGCARTC
Tn
A LI
NO
183
(HS6 (2) )
GCCCAGCCGGCCATGGCGCAGGTYCARCTGCAGCAGTC
SEQ
ID
NO
184
(HS7 (1) )
GCCCAGCCGGCCATGGCGCAGGTCCACGTGAAGCAGTC
SEQ
ID
NO
185
(HS8 <2> )
GCCCAGCCGGCCATGGCGGAGGTGAASSTGGTGGAATC
SEQ
ID
NO
186
(HS9 (5) )
GCCCAGCCGGCCATGGCGGAVGTGAWGYTGGTGGAGTC
SEQ
ID
NO
187
(HS10 (2) )
GCCCAGCCGGCCATGGCGGAGGTGCAGSKGGTGGAGTC
SEQ
ID
NO
188
(HS11 (2) )
GCCCAGCCGGCCATGGCGGAKGTGCAMCTGGTGGAGTC
SEQ
ID
NO
189
(HS12 (2> )
GCCCAGCCGGCCATGGCGGAGGTGAAGCTGATGGARTC
SEQ
ID
NO
190
(HS13 ( 1 ) )
GCCCAGCCGGCCATGGCGGAGGTGCARCTTGTTGAGTC
SEQ
ID
NO
191
(HS14 <2) )
GCCCAGCCGGCCATGGCGGARGTRAAGCTTCTCGAGTC
SEQ
ID
NO
192
(HS15 (2) )
GCCCAGCCGGCCATGGCGGAAGTGAARSTTGAGGAGTC
SEQ
ID
NO
193
(HS16 (5) )
GCCCAGCCGGCCATGGCGCAGGTTACTCTRAAAGWGTSTG
SEQ
ID
NO
194
(HS 1 7 < 3 . 5 ) ) GCCCAGCCGGCCATGGCGCAGGTCCAACTVCAGCARCC
SEQ
ID
NO
195
(HS 1 8 ( 0 . 7 ) ) GCCCAGCCGGCCATGGCGGATGTGAACTTGG AAGTGTC
SEQ
ID
NO
196
(HS19(0.7)) GCCCAGCCGGCCATGGCGGAGGTGAAGGTCATCGAGTC
SEQ
ID
NO
197
(HAKD)
GGAGCCGCCGCCGCCCGAGGAAACGGTGACCGTGGT
SEQ
ID
NO
198
(HA2 (1) )
GGAGCCGCCGCCGCCCGAGGAGACTGTGAGAGTGGT
SEQ
ID
NO
199
(HA3 ( 1 > )
GGAGCCGCCGCCGCCCGCAGAGACAGTGACCAGAGT
SEQ
ID
NO
200
(HA4 ( 1 ) )
GGAGCCGCCGCCGCCCGAGGAGACGGTGACTGAGGT
SEQ
ID
NO
201
(LSI (1) )
GGCGGCGGCGGCTCCGAYATCCAGCTGACTCAGCC
SEQ
ID
no : 202
(LS2 (2) )
GGCGGCGGCGGCTCCGA YATTGTTCTCWCCCAGTC
SEQ
ID
NO
203
(L.S3 (5) )
GGCGGCGGCGGCTCCGA YATTGTGMTMACTCAGTC
SEQ
ID
NO
204
(LS4 (3.5)) GGCGGCGGCGGCTCCGAYATTGTGYTRACACAGTC
SEQ
ID
NO
205
(LS5 (4) )
GGCGGCGGCGGCTCCGAYATTGTRATGACMCAGTC
SEQ
ID
NO
206
(LS6 (7) )
GGCGGCGGCGGCTCCGAYATTMAGATRAMCCAGTC
SEQ
ID
NO ; 207
(LS7 (6) )
GGCGGCGGCGGCTCCGAYATTCAGATGAYDCAGTC
SEQ
ID
NO: 208
(LS 8 ( 1 . 5 ) ) GGCGGCGGCGGCTCCGA YATYCAGATGACACAGAC
SEQ
ID
NO
209
(LS9 (2))
GGCGGCGGCGGCTCCGAYATTGTTCTCAWCCAGTC
SEQ
ID
NO
210
(LS 1 0 ( 3 . 5 ) ) GGCGGCGGCGGCTCCGAYATTGWGCTS ACCCAATC
SEQ
ID
NO
211
(LS11 (8) )
GGCGGCGGCGGCTCCGAYATTSTRATGACCCARTC
SEQ
ID
NO
212
(LS12 (8))
GGCGGCGGCGGCTCCGA YRTTKTGATGACCCARAC
SEQ
ID
NO
213
(LSI3 <5>)
GGCGGCGGCGGCTCCGA YATTGTGATGACBCAGKC
SEQ
ID
NO
214
(LS14 (2) )
GGCGGCGGCGGCTCCGA YATTGTGATAACYCAGGA
SEQ
ID
NO
215
(LSI5 (2))
GGCGGCGGCGGCTCCGAYATTGTGATGACCCAGWT
SEQ
ID
NO
216
(LS16 (1) )
GGCGGCGGCGGCTCCGA YATTGTGATGACACAACC
SEQ
ID
NO
217
(LS17 (1)}
GGCGGCGGCGGCTCCGA YATTTTGCTGACTCAGTC
SEQ
ID
NO
218
(LSlambda ( 1 ) ) GGCGGCGGCGGCTCCGATGCTGTTGTGACTCAGGAATC
SEQ
ID
NO
219
(LAI (4))
GGAATTCGGCCCCCGAGGCCTTGATTTCCAGCTTGG
SEQ
ID
NO
220
(L.A 2 (4) )
GGAATTCGGCCCCCGAGGCCTTTATTTCCAGCTTGG
SEQ
ID
NO
221
(LA4 (4) )
GGAATTCGGCCCCCGAGGCCTTTATTTCCAACTTTG
SEQ
ID
NO
222
(LAS (4))
GGAATTCGGCCCCCGAGGCCTTCAGCTCCAGCTTGG
SEQ
ID
NO
223
(LAlambda ( 1 ) ) GGAATTCGGCCCCCGAGGCCCCTAGGACAGTCAGTTTGG
78
3.2 Establishment of autonomously replicating cell lines by the AGS
method
The resulting library was transfected into a packaging cell,
Pt-E, (Morita, S. et al, f Gene therapy 7, 1063-1066 (2003)) using
5 FuGENE 6 (Roche Diagnostics) . Specifically, Pt-E was plated onto
6-cm dishes and cultured in DMEM/10% FBS (Invitrogen) . A mixture of
FuGENE 6 and the library was added to the plate the following day.
The culture medium was exchanged the next day, and the culture
supernatant was collected 24 hours after that. 10 ug/mL polybrene
10 (Hexadimethrine Bromide; Sigma) and 2 ng/mL mIL-3 were added to the
culture supernatant containing recombinant virus particles. The
viral solution was used to infect the BaF3-monkey Mpl target cells.
The cells were washed with PBS the following day, and suspended in
RPMI 1640/10% FBS minus mIL-3. The suspension was plated onto a
15 96-well plate at a cell density of 1,000 cells/well. Autonomously
replicating cell lines (AB317 and AB324) were obtained after seven
days of incubation. Genomic DNAs were extracted from these cells
using a DNeasy Tissue Kit (QIAGEN), and the antibody genes were
amplified by PCR.
20 The composition of the PCR reaction solution (50 uL in total)
is shown below.
lOx LA Taq Buffer (TaKaRa)
5 UL
2 mM dNTPs (dATP, dGTP, dCTP, and dTTP) (TaKaRa)
5 uL
2.5 mM MgCl 4 (TaKaRa)
5 uL
TaKaRa LA Tag (TaKaRa)
0
.5 uL
Genomic DNA
0
.5 ug
AGSdbSl (SEQ ID NO: 226) and AGSdbAl (SEQ ID NO:
227)
25
pmol
The reaction conditions were:
94 °C (initial temperature) for 1 min;
25 30 cycles of 94°C for 30 sec, 60°C for 30 sec, and 7Q°C for 1 min;
and final extension was at 72°C for 6 min.
The nucleotide sequences and the amino acid sequences of the
fragments of cloned antibodies are shown below.
80
expression vectors carrying the Mpl gene mutations were constructed
and introduced into BaF3 cells. The following Mpl gene fragments were
constructed.
Fragment Nucleotide Amino acid
sequence sequence
Normal Mpl gene SEQ ID NO: 246 SEQ ID NO: 123
Mutant Mpl gene, G305C, SEQ ID NO: 247 SEQ ID NO: 248
in which C is substituted for
305th nucleotide G relative to
the initiation codon
Mutant Mpl gene, C769T, SEQ ID NO: 249 SEQ ID NO: 250
in which T is substituted for
769th nucleotide C
Mutant Mpl gene, C823A, SEQ ID NO: 251 SEQ ID NO: 252
in which A is substituted for
823rd nucleotide C
5 The above-described DNA fragments were digested with EcoRI and Sail,
and inserted between the EcoRI and Sail sites on the animal cell
expression vector pC0S2-Ha to prepare pC0S2-hMPLf ullG305C ,
pCOS2-hMPLfullC769T, and pC0S2-hMPLfullC823A.
The genes were introduced into BaF3 cells by the procedure
10 described in Example 1 . 1 to establish BaF3 cell lines expressing each
Mpl gene: BaF3-human MPL (G305C) , BaF3-human MPL (C769T), and
BaF3-human MPL (C823A) . After the selection, the cells were cultured
and passaged using RPMI 1640 containing 1 ng/mL mIL-3 and 10% FBS .
15 4.2 Preparation of anti-human Mpl diabody and sc(Fv)2
Among the amino acid sequences shown in Figs . 6 and 7 , expression
vectors were prepared for the diabodies VB8B, VB45B, VB33, VB140,
VB157, and TA136 using the same procedure described in Example 2.2.
The prepared expression vectors were introduced into COS7 cells by
20 the same procedure described in Example 2.4. The supernatant
concentration of each diabody was determined by the method of Example
2.5. The sc (Fv) 2 expression vector for TA136 was prepared by the same
procedure described in Example 2.3. The vector was introduced into
81
CHO-DG44 cells by the same procedure described in Example 2.4.
sc ( Fv) 2 was purified from the culture supernatant thus obtained using
the same method described in Example 2.6.
5 4.3 Agonistic activity assays of sc(Fv)2 and the anti-human Mpl
diabodies
The prepared diabodies and sc(Fv)2 were assayed for their
agonistic activities in normal Mpl and mutant Mpl in BaF3 cells by
the same procedure described in Example 2.8. The agonistic
10 activities in BaF3-human Mpl and BaF3-human Mpl (G305C) were compared
using the culture supernatants of cells expressing the diabodies.
The TA136 diabody (TA136 db) was shown to have a low agonistic activity
in BaF3-human Mpl cells expressing the normal Mpl gene, and a high
agonistic activity in BaF3-human Mpl (G305C) cells expressing the
15 mutant Mpl gene. hTPO and the rest of the diabodies did not show a
high agonistic activity in BaF3-human Mpl (G305C) cells (Figs. 12
and 13) .
In addition, the agonistic activities of the TA136 diabody and
TA136 sc(Fv)2 in BaF3-human Mpl, BaF3-human Mpl (G305C) , BaF3-human
20 Mpl (C769T), and BaF3-human Mpl (C823A) cells were assessed using
a purified sample of the diabody. Compared with hTPO and the TA136
diabody, TA136 sc(Fv)2 exhibited a higher agonistic activity in all
three types of the TPO receptor mutant cell lines (Figs. 15, 16 and
17) . Furthermore, it was shown that in BaF3-human Mpl cells
25 expressing the normal Mpl gene, the TA136 diabody exhibited a lower
activity than hTPO. However, an agonistic activity equivalent to
that of hTPO was achieved by converting the diabody into sc (Fv) 2 (Fig .
14) .
30 Industrial Applicability
Various clinical trials had been conducted on recombinant human
TPO as a therapeutic agent for thrombocytopenia following
chemotherapy. Some clinical trials reported a serious problem,
namely, the production of anti-TPO antibodies due to TPO
35 administration (Junzhi Li, et al., Blood 98, 3241-324 (2001); Saroj
Vandhan-Ra j . et al., Ann. Intern. Med. 132, 364-368 (2000)).
79
Fragment
Nucleotide sequence
Amino acid
sequence
AB317
H chain
SEQ ID NO:
154
SEQ ID NO: 155
L chain
SEQ ID NO:
156
SEQ ID NO: 157
AB324
H chain
SEQ ID NO:
158
SEQ ID NO: 159
L chain
SEQ ID NO:
160
SEQ ID NO: 161
3.3 Activity assays of the diabodies obtained by AGS method
Each of the anti-Mpl diabodies obtained above was inserted into
the pCXND3 expression vector. The PCR primers used are a synthetic
5 oligonucleotide complementary to the 5' end of the diabody and
containing an EcoRI site, and a synthetic oligonucleotide
complementary to the nucleotide sequence of the 3' end of the diabody
and containing a FLAG tag and a NotI site. The PCR product thus
obtained was inserted into pCXND3 between the EcoRI and NotI sites.
10 The diabody was expressed transiently in COS7 cells by the method
described in Example 2.4. The culture supernatant was removed and
the activity of the diabody was evaluated.
The binding activities of the diabodies were assessed by flow
cytometry using CHO cells that express Mpl derived from various
15 species (Fig. 8). AB317 was proven to bind to CHO-mouse Mpl.
The TPO-like agonistic activities of the diabodies were
evaluated using BaF3-human Mpl, BaF3-monkey Mpl, and BaF3-mouse Mpl
(Figs. 9, 10, and 11). AB317 had the highest agonistic activity
against human, monkey, and mouse Mpl, whereas AB324 showed the highest
20 agonistic activity against human and monkey Mpl.
This proves that anti-Mpl diabodies having high agonistic
activity can be obtained by the AGS method.
[Example 4J Agonistic activity assays of the anti-Mpl antibodies
25 against mutant Mpl in congenital amegakaryocyt ic thrombocytopenia
(CAMT) patients
4.1 Establishment of BaF3 cell lines introduced with the mutant Mpl
observed in CAMT patients
Mutations on G305C (R102P), C769T (R257C) , and C823A (P275T)
30 have been reported in the Mpl gene of CAMT patients. The respective
82
Specifically, the production of neutralizing antibodies which inhibit
the activity of endogenous TPO have been reported, triggering the
onset of thrombocytopenia. In the present invention, the
administration of agonistic minibodies against TPO receptor does not
5 induce the production of antibodies against endogenous TPO.
Reduction of the molecular weight of antibodies increases the specific
activity of antibodies and shortens the half-life in blood. Thus,
the effective concentration of an antibody in blood can be easily
controlled, presenting an advantage in clinical applications.
10 Accordingly, such an antibody can be used as an agent to treat
thrombocytopenia more effectively than the naturally-occurring TPO
or its agonistic antibodies. Since minibodies are not attached with
sugar chains, the expression systems for expressing those recombinant
proteins are not limited, and minibodies can be prepared by using
15 any of the expression systems derived from mammalian cell lines,
yeasts, insect cells, and E. coli. In addition, minibodies have a
binding affinity towards mutant TPO receptor different from that of
TPO. Therefore, minibodies are expected to bind and exhibit
agonistic activities against specific TPO receptor mutants, which
20 contain mutations commonly detected in CAMT patients with
thrombocytopenia and genetically mutated TPO receptors.
83
CLAIMS
1. An antibody comprising a single-chain polypeptide having binding
activity against TPO receptor (Mpl), wherein said antibody comprises
5 two heavy chain variable regions and two light chain variable regions.
2. The antibody of claim 1, wherein the two heavy chain variable
regions and the two light chain variable regions are arranged in the
order of heavy chain variable region, light chain variable region,
10 heavy chain variable region, and light chain variable region from
the N terminus of the single-chain polypeptide.
3. The antibody of claim 1 or 2, wherein the two heavy chain variable
regions and the two light chain variable regions are linked by linkers .
15
4. The antibody of claim 3, wherein the linkers comprise 15 amino
acids .
5. A chimeric antibody that binds to Mpl.
20
6. The antibody of claim 5, which is a humanized antibody.
7. The antibody of claim 5 or 6, which is a minibody.
25 8 . An antibody that binds to soluble Mpl.
9. An antibody that binds to human Mpl and monkey Mpl.
10. An antibody having agonistic activity against human Mpl and monkey
30 Mpl.
11. An antibody whose binding activity to soluble Mpl is KD = 10" 6
M or lower.
35
12. An antibody whose binding activity to soluble Mpl is KD = 10
M or lower.
84
13. An antibody whose TPO agonistic activity is EC50 = 100 nM or lower.
14. An antibody whose TPO agonistic activity is EC50 = 30 nM or lower.
5
15. An antibody whose TPO agonistic activity is EC50 = 10 nM or lower.
16. An antibody which comprises a heavy chain variable region, wherein
said heavy chain variable regions comprises CDR1, CDR2 and CDR3
10 consisting of an amino acid sequence of any one of:
[1] SEQ ID NOs: 3, 4, and 5
[2] SEQ ID NOs: 6, 7, and 8
13] SEQ ID NOs: 9, 10, and 11
[4] SEQ ID NOs: 15, 16, and 17
15 [5] SEQ ID NOs: 18, 19, and 20
(6] SEQ ID NOs: 21, 22, and 23
(7] SEQ ID NOs: 24, 25, and 26
[8] SEQ ID NOs: 27, 28, and 2 9
[9]
SEQ ID NOs:
30,
31,
and
32
20
[10]
SEQ
ID
NOs:
33,
34,
and
35
[11]
SEQ
ID
NOs:
36,
37,
and
38
[12]
SEQ
ID
NOs:
39,
40,
and
41
[13]
SEQ
ID
NOs:
42,
43,
and
44
[14]
SEQ
ID
NOs:
48,
49,
and
50
25
[15]
SEQ
ID
NOs:
51,
52,
and
53
[16]
SEQ
ID
NOs:
54,
55,
and
56
[17]
SEQ
ID
NOs:
57,
58,
and
59.
17 . An antibody which comprises a light chain variable region, wherein
30 said light chain variable region comprises CDR1, CDR2 and CDR3
consisting of an amino acid sequence of any one of:
[1] SEQ ID NOs: 60, 61, and 62
[2] SEQ ID NOs: 63, 64, and 65
[3] SEQ ID NOs: 78, 79, and 80
35 [4] SEQ ID NOs: 84, 85, and 86
[5] SEQ ID NOs: 93, 94, and 95
85
[6] SEQ ID NOs: 96, 97, and 98
[7] SEQ ID NOs: 102, 103, and 104
[8] SEQ ID NOs : 108, 109, and 110
[9] SEQ ID NOs: 111, 112, and 113
[10] SEQ ID NOs: 114, 115, and 116.
18. An antibody that comprises a heavy chain variable region and a
light chain variable region of any one of:
[1] a heavy chain variable region that comprises CDR1 , CDR2 ,
and CDR3 comprising the amino acid sequences consisting of SEQ ID
NOs: 3, 4, and 5 r and a light chain variable region that comprises
CDR1, CDR2, and CDR3 comprising the amino acid sequences consisting
of SEQ ID NOs: 60, 61, and 62;
[2] a heavy chain variable region that comprises CDR1, CDR2,
and CDR3 comprising the amino acid sequences consisting of SEQ ID
NOs: 6, 7, and 8, and a light chain variable region that comprises
CDR1, CDR2 , and CDR3 comprising the amino acid sequences consisting
of SEQ ID NOs: 63, 64, and 65;
[3] a heavy chain variable region that comprises CDR1, CDR2,
and CDR3 comprising the amino acid sequences consisting of SEQ ID
NOs: 9, 10, and 11, and a light chain variable region that comprises
CDR1, CDR2, and CDR3 comprising the amino acid sequences consisting
of SEQ ID NOs: 63, 64, and 65;
[4] a heavy chain variable region that comprises CDR1, CDR2,
and CDR3 comprising the amino acid sequences consisting of SEQ ID
NOs: 15, 16, and 17, and a light chain variable region that comprises
CDR1, CDR2, and CDR3 comprising the amino acid sequences consisting
of SEQ ID NOs: 63, 64, and 65;
[5] a heavy chain variable region that comprises CDR1, CDR2,
and CDR3 comprising the amino acid sequences consisting of SEQ ID
NOs: 18, 19, and 20, and a light chain variable region that comprises
CDR1, CDR2, and CDR3 comprising the amino acid sequences consisting
of SEQ ID NOs: 63, 64, and 65;
[6] a heavy chain variable region that comprises CDR1, CDR2,
and CDR3 comprising the amino acid sequences consisting of SEQ ID
NOs: 21, 22, and 23, and a light chain variable region that comprises
86
CDR1, CDR2, and CDR3 comprising the amino acid sequences consisting
of SEQ ID NOs: 78, 79, and 80;
[7] a heavy chain variable region that comprises CDRl, CDR2 ,
and CDR3 comprising the amino acid sequences consisting of SEQ ID
NOs: 24, 25, and 26, and a light chain variable region that comprises
CDRl, CDR2, and CDR3 comprising the amino acid sequences consisting
of SEQ ID NOs: 63, 64, and 65;
[8] a heavy chain variable region that comprises CDRl, CDR2,
and CDR3 comprising the amino acid sequences consisting of SEQ ID
NOs: 27, 28, and 29, and a light chain variable region that comprises
CDRl, CDR2, and CDR3 comprising the amino acid sequences consisting
of SEQ ID NOs: 84, 85, and 86;
[9] a heavy chain variable region that comprises CDRl, CDR2,
and CDR3 comprising the amino acid sequences consisting of SEQ ID
NOs: 30, 31, and 32, and a light chain variable region that comprises
CDRl , CDR2, and CDR3 comprising the amino acid sequence consisting
of SEQ ID NOs: 63, 64, and 65;
[10] a heavy chain variable region that comprises CDRl, CDR2,
and CDR3 comprising the amino acid sequences consisting of SEQ ID
NOs: 33, 34, and 35, and a light chain variable region that comprises
CDRl, CDR2, and CDR3 comprising the amino acid sequences consisting
of SEQ ID NOs: 63, 64, and 65;
[11] a heavy chain variable region that comprises CDRl, CDR2,
and CDR3 comprising the amino acid sequences consisting of SEQ ID
NOs: 36, 37, and 38, and a light chain variable region that comprises
CDRl, CDR2 , and CDR3 comprising the amino acid sequences consisting
of SEQ ID NOs: 93, 94, and 95;
[12] a heavy chain variable region that comprises CDRl, CDR2,
and CDR3 comprising the amino acid sequences consisting of SEQ ID
NOs: 39, 40, and 41, and a light chain variable region that comprises
CDRl, CDR2, and CDR3 comprising the amino acid sequences consisting
of SEQ ID NOs: 96, 97, and 98;
[13] a heavy chain variable region that comprises CDRl, CDR2,
and CDR3 comprising the amino acid sequences consisting of SEQ ID
NOs: 42, 43, and 44, and a light chain variable region that comprises
CDRl, CDR2, and CDR3 comprising the amino acid sequences consisting
87
of SEQ ID NOs: 78, 79, and 80;
[14] a heavy chain variable region that comprises CDR1, CDR2,
and CDR3 comprising the amino acid sequences consisting of SEQ ID
NOs: 45, 46, and 47, and a light chain variable region that comprises
CDRl, CDR2, and CDR3 comprising the amino acid sequences consisting
of SEQ ID NOs: 102, 103, and 104;
[15] a heavy chain variable region that comprises CDRl, CDR2,
and CDR3 comprising the amino acid sequences consisting of SEQ ID
NOs: 48, 49, and 50, and a light chain variable region that comprises
CDRl, CDR2, and CDR3 comprising the amino acid sequences consisting
of SEQ ID NOs: 63, 64, and 65;
[16] a heavy chain variable region that comprises CDRl, CDR2,
and CDR3 comprising the amino acid sequences consisting of SEQ ID
NOs: 51, 52, and 53, and a light chain variable region that comprises
CDRl , CDR2, and CDR3 comprising the amino acid sequences consisting
of SEQ ID NOs: 108, 109, and 110,
[17] a heavy chain variable region that comprises CDRl, CDR2 ,
and CDR3 comprising the amino acid sequences consisting of SEQ ID
NOs: 54, 55, and 56, and a light chain variable region that comprises
CDRl, CDR2, and CDR3 comprising the amino acid sequences consisting
of SEQ ID NOs: 111, 112, and 113;
[IB] a heavy chain variable region that comprises CDRl, CDR2,
and CDR3 comprising the amino acid sequences consisting of SEQ ID
NOs: 57, 58, and 59, and a light chain variable region that comprises
CDRl, CDR2, and CDR3 each comprising the amino acid sequences
consisting of SEQ ID NOs: 114, 115, and 116.
19. An antibody that comprises a heavy chain variable region
comprising the amino acid sequence of SEQ ID NO: 118.
20. An antibody that comprises a light chain variable region
comprising the amino acid sequence of SEQ ID NO: 120.
21. An antibody that comprises a heavy chain variable region
comprising the amino acid sequence of SEQ ID NO: 118 and a light chain
variable region comprising the amino acid sequence of SEQ ID NO: 120.
88
22. An antibody comprising the amino acid sequence of SEQ ID NO: 122
or 264.
2 3. An antibody that comprises a heavy chain variable region, wherein
said heavy chain variable region comprises FR1, FR2, FR3, and FR4
consisting of amino acid sequences of any one of:
[1] SEQ ID NOs: 230, 232, 234, and 236
[2] SEQ ID NOs: 265, 267, 269, and 271
[3] SEQ ID NOs: 279, 281, 283, and 285
[4] SEQ ID NOs: 298, 299, 300, and 301
[5] SEQ ID NOs: 298, 299, 306, and 301.
24. An antibody comprising a light chain variable region, wherein
said light chain variable region comprises FR1, FR2, FR3, and FR4
consisting of amino acid sequences of any one of:
[1] SEQ ID NOs: 239, 241, 243, and 245
[2] SEQ ID NOs: 272, 274, 276, and 278
[3] SEQ ID NOs: 302, 303, 304, and 305
[4] SEQ ID NOs : 302, 307, 308, and 305.
25. An antibody that comprises a heavy chain variable region and a
light chain variable region of any one of:
[1] a heavy chain variable region which comprises FR1 , FR2 , FR3,
and FR4 having the amino acid sequences consisting of SEQ ID NOs:
230, 232, 234, and 236, and a light chain variable region which
comprises FR1, FR2, FR3, and FR<3 having the amino acid sequences
consisting of SEQ ID NOs: 239, 241, 243, and 245;
[2] a heavy chain variable region which comprises FR1, FR2, FR3,
and FR4 having the amino acid sequences consisting of SEQ ID NOs:
265, 267, 269, and 271, and a light chain variable region which
comprises FR1, FR2, FR3, and FR4 having the amino acid sequences
consisting of SEQ ID NOs: 272, 274, 276, and 278;
[3] a heavy chain variable region which comprises FR1 , FR2 , FR3,
and FR4 having the amino acid sequences consisting of SEQ ID NOs:
279, 281, 283, and 285, and a light chain variable region which
89
comprises FR1, FR2, FR3 P and FR4 having the amino acid sequences
consisting of SEQ ID NOs: 272, 274, 276, and 278;
[4] a heavy chain variable region which comprises FR1, FR2, FR3,
and FR4 having the amino acid sequences consisting of SEQ ID NOs:
298, 299, 300, and 301, and a light chain variable region which
comprises FR1 , FR2 , FR3, and FR4 having the amino acid sequences
consisting of SEQ ID NOs: 302, 303, 304, and 305;
[5] a heavy chain variable region which comprises FR1 , FR2, FR3,
and FR4 having the amino acid sequences consisting of SEQ ID NOs:
298, 299, 306, and 301, and a light chain variable region which
comprises FR1, FR2, FR3, and FR4 having the amino acid sequences
consisting of SEQ ID NOs: 302, 307, 308, and 305.
26. An antibody that comprises a heavy chain variable region, wherein
said heavy chain variable region comprises the amino acid sequence
of SEQ ID NO: 229, 256, 262, 289, or 295.
27. An antibody that comprises a light chain variable region, wherein
said light chain variable region comprises the amino acid sequence
of SEQ ID NO: 238, 258, 291, or 297.
28. An antibody that comprises a heavy chain variable region and a
light chain variable region of any one of:
[1] a heavy chain variable region comprising the amino acid
sequence of SEQ ID NO: 229, and a light chain variable region
comprising the amino acid sequence of SEQ ID NO: 238;
[2] a heavy chain variable region comprising the amino acid
sequence of SEQ ID NO: 256, and a light chain variable region
comprising the amino acid sequence of SEQ ID NO: 258;
[3] a heavy chain variable region comprising the amino acid
sequence of SEQ ID NO: 262, and a light chain variable region
comprising the amino acid sequence of SEQ ID NO: 258;
[4] a heavy chain variable region comprising the amino acid
sequence of SEQ ID NO: 289, and a light chain variable region
comprising the amino acid sequence of SEQ ID NO: 291;
[5] a heavy chain variable region comprising the amino acid
90
sequence of SEQ ID NO: 295, and a light chain variable region
comprising the amino acid sequence of SEQ ID NO: 297.
29. An antibody that comprises the amino acid sequence of SEQ ID NO:
2, 254, 260, 287, or 293.
30. An antibody having an activity equivalent to that of an antibody
of any one of claims 16 to 29, wherein said antibody comprises the
amino acid sequence set forth in any one of claims 16 to 29, in which
one or more amino acids have been substituted, deleted, added and/or
inserted .
31. An antibody that recognizes an epitope recognized by an antibody
of any one of claims 16 to 30.
32. An antibody that recognizes the region of amino acids 26 to 274
of human Mpl .
33. An antibody of any one of claims 1 to 32, which has TPO agonistic
activity.
34. A polynucleotide encoding an antibody of any one of claims 1 to
33.
35. A polynucleotide hybridizing to the polynucleotide of claim 34
under stringent conditions, wherein said polynucleotide encodes an
antibody having activity equivalent to that of an antibody of any
one of claims 1 to 33.
36. A vector comprising the polynucleotide of claim 34 or 35.
37. A host cell that carries the polynucleotide of claim 34 or 35,
or the vector of claim 36.
38. A pharmaceutical composition comprising an antibody of any one
of claims 1 to 33.
91
ABSTRACT
Anti-human Mpl antibodies were isolated and purified, and then
anti-human Mpl diabodies and anti-human Mpl sc(Fv)2 were purified
using genetic engineering techniques. Furthermore, the present
inventors succeeded in humanizing anti-human Mpl sc(Fv)2.
The diabodies and sc(Fv)2 were assayed for TPO-like agonistic
activity, and were found to have activities higher than those of
anti-human Mpl antibodies, or activities equivalent to or higher than
those of naturally-occurring human TPO ligand.
1/22
FIG.
1
2/22
CHO-HUMAN Mpl
i'o° 101 ' ' "'163
FITC LOG
CHO-MOUSE Mpl
lbo io< ' lbs 103
FITC LOG
CHO-MONKEY Mpl
FITC LOG
FIG. 2
FIG. 3
4/22
1.8
0.000 0.001 0.010 0.100 1.000 10.000
CONCENTRATION [nM]
FIG. 4
5/22
1.4
0 i ' 1 ' 1 1 ■■ 1
0.0001 0.001 0.01 0.1 1 10 100
CONCENTRATION (nM)
FIG. 5
6/22
VA7
VA130
VA259
VD17B
VB12B
VB140
VB33
VB4SB
VB8B
VB11S
VB14B
VB72B
VB16
VB157
VB4B
VB51
VA7
VA130
VA2 59
VB17B
VB12B
VB140
VB33
VB4 5B
VB8B
V8115
VB14B
VB22B
VB16
VB157
VB4B
VB51
QVQLQQ S G ?E L VK PG AS VK I S CKAS G YA FS
QVQLQQSGPELVKPGASVKI SCKASGYAFS
QVQLQQSGPELVKPGASVKISCKASGYAFS
QVQLQQSGPELVKPGASVKI SCKASGYTFS
QVQLQQSGPELVKPGASVKI SCKASGYAFS
QVQLQQSGFELVKPGASVKISCRAFGYAFS
QVQLQQPGAELVKPGAS VKLSCKASGYT FT
QVQLQQSGPELVKPGASVKISCKASGYAFS
QVQLQQSGPELVKPGASVKI SCKASGYAFS
QVQLQQSGPELVKPGAS VKI SCKASGYAFS
QVQLQQSGPELLNPGASVKISCKASGYAFS
QVQT.QQSGPF.LVKPGARVKTSCKASGYAFT
QVQLQQPGTELVRPGAS VKLSCKASGYT FT
QVQLQQPGAELVKPGAS VKLSCKASGYT FT
QVQLQQSC PELVKPGAS VKI S CKASGYAFT
QVQLQQS GPELVKPGAS VKI SCKASGYAFS
CDR1
SSWMN WVKQRPGKGLEWIG
SSWMN WVKQRPGKGLEWIG
SSWMN WVKQRPGKGLEWIG
SSWMN WVKQRPGKGLEWIG
RSWMN WVKQRPGKGLEWIG
NSWMN WVKQRPGKGLEWIG
NYWVN WVKQR PGRGLEW I G
SSWMN WVKQRPGKGLEWIG
TSWMN WVKQRPGKGLEWIG
SSWMN WVKQR PGKGPEWIG
RSWMN WVKQR PGKGLEWIG
NSWMN WVKQR PRKG1.F.WIG
DYWVN WVKQRPGRGLEWIG
DYWMN WVKQRPGRGLEWIG
NSWMN WV3QRPCKCLEWIG
NSWMN WVNQRPGKGLEWIG
CDR3
CDR2
RTYPGDGDTNYNGKFKG
RIYPGDGDTNYNGKFKG
RIYPGDGETNYNGKFKG
RIYPGDGDTNYNGKFKG
RIYPGDGDTNYNGKFKG
RIYPGDGETNNNGKFKG
RIHPSDSETHCNQKFKR
RIYPGDGETNNNGKFKG
RIYPGDGEANYNGKFKG
RIYPGDGETNYNGKFKG
RIYPGDGETNYNGKFKG
RTYPGDGF.TT YNGKFRV
RIHPYDSETHYNQKFKN
RIHPFDSETHCSQKFKN
RIYPGDCETIYNCKFRV
RI YPGDGDTI YNGNFKG
KATLTADKSSSTAYMQLSSLTSEDSAVYFCAR
KATLTADKSSSTAYI QLSSLTSEDSAVY FCAR
KATLTADKSSWTAYMQLSSLTSEDSAVY FCAR
KATLTADKSSSTAYMQLSSLTSEDSAVY FCAS
KATLTADKS5STAYMQLSSLTSEDSAVYFCA5
KATLTADKSSSTAYMQLSSLTSEDSAVY FCAR
KATLTVNKSSSTAYIQLHSLTSEDSAVYYCTS
KATLTADKSS7TAYMQLSSLTSEDSAVY FCAR
KATLTADKSSSSAYMQLSSLTSEDSAVYFCAR
KATLTADKSSSTVYMQLSSLTSEDSAVY FCAR
KAT LTADKSSCTAYMQFSSLTSEDSAVY FCAR
KATLTADKSSSTAYMDISSLTSEDSAVYFCAR
KATL'J'VDKSS STAY! QLSSLTSEDSAVY YCAS
KATLTVDKSSNTAYIQFSSLTSEDSAVY YC3S
KATLTADKSSSTAYMEISSLTSEDSAVY FCAR
KATLTADKSSSI AYMQLSSLTSEDSAVY FCTS
GWILADGGYS
FAY
WGQGTLVTVSA
(SEQ ID NO:
124 )
GYAD--
— YS
FAY
WCQCTLVTVSA
( SEQ ID NO:
126)
GFGD —
— YS
FAY
WGQGTLVTVSA
( SEQ ID NO:
12B)
GYAD--
— YS
FAY
WGQGTLVTVSA
( SEQ 10 NO:
130)
GYDD —
— YS
FAY
WGQGTLVTVSA
(SEQ ID NO.
132)
GYGD--
— YS
FAY
WGQGTLVTVSA
(SEQ ID NO
111 )
GGW
FAY
WGQGTLVTVSA
( SEQ ID NO:
136)
GYGD--
— YS
FAY
WGQGTLVTVSA
( SEQ 10 NO:
138)
GYGD--
--YS
FAY
WGQGTLVTVSA
(SEQ ID NO:
140)
GYGD--
— YS
FAY
WGQGTLVTVSA
( SEQ ID NO:
142)
GDGD--
— YS
FAY
WGQGTLVTVSA
( SEQ ID NO:
144 )
GYDD--
— YS
FAY
WGQGTLVTVSA
( SEQ ID NO:
118)
GGW
FAS
WGQGTLVTVSA
( SEQ ID NO
146)
GGW
FAY
WGQGTLVTVSA
(SEQ ID NO
148)
GYDD--
--YS
FAY
WGQGTLVTVSA
( SEQ ID NO
150)
GYDD--
--YS
FAY
WGQGTLVTVSA
( SEQ ID NO
152)
FIG. 6
7/22
VA7
VA130
VA259
VB17B
VB12B
VB140
VB33
VB4 5B
VB83
VB11S
VB14B
VB22B
VB1 6
VB1S7
VB43
VBS1
VA7
VA130
VA259
VB17B
VB12B
VB140
VB33
VB4 5B
VB83
VB115
VB14B
VB22B
VB16
VB1 57
VB4 3
VB51
D I VMTQAAPS I PVT PGES VS ISC
DIVMTQAAPSVPVTPGESVSISC
DIVMTQAAPSVPVTPGESVSISC
D IVNTQAAPSVPVT PGESVS ISC
D IVMTQAAPSVPVT PGESVS ISC
DIVMTQAAPSVPVTPGESVSISC
DIVMTQAAPSVPVTPGESVSISC
DIVMTQAAPSVPVTPGESVSISC
DIVMTQAAPSVPVTPGESVSISC
DIVMTQAAPSVPVTPGESVSISC
D IVMTQAAPSVPVT PGESVS ISC
D I VMTQAAPS I PVTPGES VS ISC
DIVMTQAAPSVPVTPGESVSISC
DIVMTQAAPSVSVTPGESVSISC
DIVMTQAAPSVPVTPGESVSISC
DIVMTQAAP5LPVTPGESVSISC
CDR1
RSSKSLLHSNGNTYLY
RSSKSLLHSNGNTYLY
RSSKSLLHSNGNTYLY
RSSKSLLHSNGNTYLY
RSSKSLLHSNGNTYLY
RSSKSLLHSNGNTYLY
RSSKSLLYSNGNI YLY
RSSKSLLHSNGNTYLY
RSSKSLLHSNGNTYLY
RSSKSLLHSNGNTYLY
RSSKSLLHSNGNTYLY
RSSKSLLHSNGNTYLY
RSSKSLLY SNGNTYLY
RSSKSLLYSNGNI YLY
RSSKSLLHNNGNTYLY
RSSKSLLHSNGNTYLY
WFLQRPGQSPQLLIY
WFLQRPGQSPQLLIY
WFLQRPGQSPQLLIY
WFLQRPGQSPQLLIY
WFLQRPGQSPQLLIY
WFLQRPGQSPQLLIY
WFLQRPGQSPQLLIY
WFLQRPGQSPQLLIY
WFMQR PGQS PQLL I Y
WFLQRPGQSPQLLIY
WFLQRPGQSPQLLIY
WFLQRPGQSPQLLIY
WFLQRPGQSPQLLIY
WFLQRPGQSPQLLIY
WFLQRPGQSPQLLIY
WFLQRPGQSPQLLIY
CDR2
RMSNLAS
RMSNLAS
RMSNLAS
RMSNLAS
RMSNLAS
RMSNLAS
RMSNLAS
RMSNLAS
RMSNLAS
RMSNLAS
RMSNLAS
RMSNLAS
RMSNLAS
RMSNLAS
RMSNLAS
RMSNLAS
CDR3
GVPDRFSGSGSGTAFTLRI
GVPDRFSGSGSGTAFTLRI
GAPDRFSGSGSG7AFTLRI
GVPDRFSGSGSGTAFTLRI
GVPDRFSGSGSGTAFTLRI
GVPDRFSGSGSGAAFTLRI
GVPDRFSGSGSGTAFTLRI
GVPDRFSGSGSGAAFTLRI
GVPDRFSGSGSGTAFTLRI
GVPDRFSGSGSGTAFTLRI
GVPDRFSGSGSGTAFTLRI
GVPDRFSGSGSGTAFTLRI
GVPDRFSGSGSGTAFTLTI
GVPDR FSGSGSGTft FTT.iKT
GVPDRFSGSGSGTAFTLRI
GVPDRFSGSGSGTAFTLRI
SRVEAEDVGI YYC
SRVEAEDVGVYYC
SRVETEDVGVYYC
SRVEAEDVGVYYC
SRVEAEDVGVYYC
SRVEAEDVGVYYC
SRVEAEDVGVYYC
SRVEAEDVGVYYC
SRVEAEDVGVYYC
SRVEAEDVGVYYC
SRVEAEDVGVYYC
SRVEAEDVGVYYC
SSVEAEDVGVYYC
5RVERKDVGVYYC
SRVEAEDVGVYYC
SRVEAEDVGVYYC
MQHLEYPFT
MQHLEYPYT
MQHLEYPYT
MQHLEYPYT
MQHLEYPYT
MQHLEYPYT
MQHLEYPYT
MQHLEYPYT
MQHVEYPYT
MQHLEYPYT
MQHLEYPYT
MQHIEYPFT
MQHLEYPYT
MQHT..F.YPYT
MQHIEYPFT
MQHLEYPYT
FGTGTKLEIK
FGSGTKLEIK
FGSGTKLEIK
FGSGTKLEIK
FGSGTKLEIK
FGSGTKLEIK
FGSGTKLEIK
FGSGTKLEIK
FGSGTKLEIK
FGSGTKLEIK
FGSGTKLEIK
FGSGTKLEIK
FGSGTKLEIK
FGSGTKTiETK
FGSGTKLEIK
FGSGTKLEIK
(SEOID
( SEO ID
(SEOID
( SEO ID
( SEO ID
( SEDID
( SEO ID
{ SEO ID
( SEQ ID
( SEQ ID
( SEQ ID
( SEQ ID
( SEQ ID
( SEQ ID
( SEQ ID
(SEQ ID
NO: 125)
NO; 127)
NO- 129 )
NO: 131 )
NO; 133)
NO: 135)
NO: 137)
NO: 139)
NO; 141 )
NO: 143)
NO: 145)
NO: 120 )
NO: 147 )
NO: 149)
NO: 151 )
NO: 153)
FIG. 7
8/22
FIG. 8
9/22
FIG. 9
10/22
FIG. 10
11/22
1
0.000 0.001 0.010 0.100 1.000 10.000 100.000
CONCENTRATION [nM]
FIG. 11
12/22
0.1
1.0 10.0 100.0
CONCENTRATION (ng/ml)
FIG. 12
♦-hTPO
*-VB08Bdb
-*-VB45Bdb
VB033db
-*-VB140db
H-VB157db
-*-TA136db
1000.0
13/22
FIG. 13
14/22
15/22
-•-TA136 sc(Fv)2
-♦-TA136 db
-*-TPO
o
o
o
o
o
o
o
o
o
o
o
o
o
o
o
o
o
CONCENTRATION (nM)
o
o
o
o
o
FIG. 15
16/22
FIG. 16
17/22
FIG. 17
18/22
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FIG. 18-2
20/22
0.000 0.001 0.010 0.100 1.000 10.000
CONCENTRATION [nM]
FIG. 19
21/22
FIG. 20
22/22
1.600
1.400
1.200
1.000
0.800
0.600
0.400
0.200
0.000
-3-VB22B
hVB22B q-wz5
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0.000 0.001 0.010 0.100 1.000
CONCENTRATION [nM]
10.000
FIG. 21
Live Music Archive
Librivox Free Audio
Metropolitan Museum
Cleveland Museum of Art
Internet Arcade
Console Living Room
Books to Borrow
Open Library
TV News
Understanding 9/11